Assembly activating protein (aap) and its use for the manufacture of parvovirus particles essentially consisting of vp3

ABSTRACT

The present invention relates to nucleic acids encoding the novel parvoviral protein “assembly activating protein” S(AAP), the encoded polypeptides, methods of producing the polypeptides, antibodies specific for AAP, the use of the nucleic acids for the preparation of the polypeptides, the use of the nucleic acids or the polypeptides for the preparation of the parvoviral particle and methods of producing parvoviral particles essentially consisting of VP3 by providing in addition to the coding sequence of the parvoviral structural protein VP3 a sequence fragment Z/a nucleic acid encoding AAP in the cell and expressing VP3 and fragment Z under control of a rep-independent promoter. Furthermore, the present invention relates to parvoviral particles essentially consisting of VP3 and/or obtainable by the above method as well as expression cassettes comprising (i) a heterologous promoter and (ii) VP3 coding sequence and/or fragment Z. The present invention further relates to a medicament, particularly a vaccine, comprising the parvoviral particles or expression cassettes and their use.

The present invention relates to nucleic acids encoding the novelparvoviral protein “assembly activating protein” (AAP), the encodedpolypeptides, methods of producing the polypeptides, antibodies specificfor AAP, the use of the nucleic acids for the preparation of thepolypeptides, the use of the nucleic acids or the polypeptides for thepreparation of the parvoviral particle and methods of producingparvoviral particles essentially consisting of VP3 by providing inaddition to the coding sequence of the parvoviral structural protein VP3a sequence fragment Z/a nucleic acid encoding AAP in the cell andexpressing VP3 and fragment Z under control of a rep-independentpromoter. Furthermore, the present invention relates to parvoviralparticles essentially consisting of VP3 and/or obtainable by the abovemethod as well as expression cassettes comprising (i) a heterologouspromoter and (ii) VP3 coding sequence and/or fragment Z. The presentinvention further relates to a medicament, particularly a vaccine,comprising the parvoviral particles or expression cassettes and theiruse.

Mutated parvovirus structural protein-based virus-like particles (VLPs)have been shown to be suitable vaccine candidates (WO 2008/145401,hereby incorporated by reference). Based on such mutated parvovirusstructural proteins, VLPs were generated for the presentation oftolerogens or small antigens or even individual epitopes. These VLPsproved especially beneficial, where B cell tolerance has to be broken tohave a therapeutic effect for the patient.

For the clinical development of vaccines based on VLPs it is generallynecessary to generate a product which ideally is based on a singleactive compound/protein and which is as pure as possible. With respectof VLPs this is a problem in general as viruses are often composed ofmore than one protein and are capable of packaging specifically viralDNA or unspecifically DNA from the host cell. Accordingly it isdesirable to obtain “pure” VLPs that contain as few different proteinsas possible and preferably no nucleic acid. In the literature, severalattempts have been made to efficiently produce those particles.

Rabinowitz et al. (e.g. Rabinowitz et al., 1999) have altered thestructural genes of AAV2 by linker insertional mutagenesis in order todefine critical components of virion assembly and infectivity. Theygenerated the mutant H2634 that contains the rep and cap ORFs and aninsertion at the HaeIII restriction site at position 2634. Importantly,due to the presence of the rep ORF this insertion mutant expressed therespective Rep protein. It assembled intact virions and the capsidappeared to be composed only of VP3. According to the authors theundetectable expression of VP1 and VP2 in either cell lysates orpurified virions could have been a problem of detection limits.

Warrington et al. (2004) and WO 2004/027019 also addressed the questionof the specific roles of the individual capsid proteins in capsidformation to define where full-length peptides can be inserted into theAAV capsid ORF without disruption of critical structural domains.Generating constructs containing the rep and cap ORF with mutations inthe start codons of VP1, VP2 and/or VP3 and thus expressing only asingle or two capsid protein(s) in the presence of Rep, Warrington etal. showed that genome-containing particles were formed as long as theVP3 protein was present. Hence, mutants expressing VP1 and/or VP2 assingle capsid proteins or together did not form particles. Rather theyconcluded from their results that VP1 is necessary for viral infectionbut not essential for capsid assembly and particle formation whereas VP2appears to be nonessential for viral infectivity. Moreover, theyobserved that expression of VP3 alone from constructs with mutated startcodons for VP1 and VP2 is sufficient to form VLPs.

Just as well, Grieger et al. (2007) generated VP3-only particles usingthe AAV2 helper plasmid pXR2 (containing rep and cap genes, Li et al.(2008)) via mutagenesis of the VP1 and VP2 start codon. Expression ofVP3- as well as VP2/VP3-only constructs in the presence of Rep resultedin noninfectious viral particles as long as they lacked the VP1 subunit.

From their results on the formation of genome-containing AAV-likeparticles from mutants expressing VP3 as only capsid protein in thepresence of Rep it seemed that these particles can readily be obtained.

All the expression constructs described above expressed Rep proteinswhich should be omitted to assemble VLPs that are composed preferably ofone protein and no DNA. Rep does not only represent a further proteinthat is attached to VLPs but also is held responsible for packaging ofvirus genomes and unspecific DNA into preformed capsids (King et al.,2001). Packaging of DNA is to be avoided as VLPs potentially can entercells of a patient and thereby transfect such contaminating DNA, whichmay cause all sorts of unwanted effects.

To be sure that only VP3 is expressed, Hoque et al. (1999a, 1999b) andHanda et al. (JP 2001169777) generated expression constructs comprisingthe coding sequence (cds) of VP3 alone under control of a heterologouspromoter in the absence of any Rep cds. Surprisingly, they could notproduce viral particles from these expression constructs. By analyzing aseries of deletion mutants of VP2 that started expression at differentsites 5′ of the VP3 start codon, they identified a region necessary fornuclear transfer of VP3 and found that the efficiency of nuclearlocalization of the capsid proteins and the efficiency of VLP formationcorrelated well. They observed that viral particles were formed as longas a region between amino acid 29 and 34 in the cds of VP2 or in otherwords in the 5′ extension of VP3, was present. From the amino acid motifof this region which is PARKRL they concluded that it functions as anuclear localization signal (NLS) which is important for thetranslocation of VP3 into the nucleus.

Alternatively, capsids also could be obtained if the NLS of simian virus40 (SV40) large T antigen was fused to the N-terminus of the VP3 protein(NLS_(SV40)-VP3). This fusion protein could form VLPs indicating thatthe VP2-specific region located on the N-terminal side of the protein isnot structurally required. Due to this finding the authors reasoned thatVP3 has sufficient information for VLP formation and that VP2 isnecessary only for nuclear transfer of the capsid proteins, which againis a prerequisite for VLP formation.

Due to the method for mutant construction used by them, all constructsstarted with an ATG start codon directly at the 5′ end of the codingsequence. Since in general the “position effect” (Kozak, 2002) willcause the first (most upstream) ATG start codon of a transcript toinitiate translation, the main protein to be expressed and generatingthe particle will be N-terminally extended VP3. Only a minor part oftranslation will start at the further downstream ATG start codon of VP3.

In agreement with Hoque et al. (supra) and Handa et al. (supra) andusing constructs described by them, we could not detect VLPs consistingof VP3 alone from expression constructs comprising the cds of VP3 aloneunder control of a constitutive promoter in neither mammalian cells norinsect cells in quantitative amounts (meaning that <10¹⁰, particularly<10⁸ capsids/ml were present) using the AAV2 Titration ELISA (quantifiedaccording to the instructions of the manufacturer Progen, Heidelberg,Germany, FIG. 15B). Nor could we detect AAV-like particles expressingVP1 or VP2 alone from expression constructs comprising the respectivecds alone starting with an ATG codon under control of a constitutivepromoter. The efficiency of capsid production of all constructs alone orin different combinations of different ratios in the presence or absenceof Rep expression and in the presence or absence of co-delivery ofadenoviral helper genes was at the lower detection limit of the AAV2Titration ELISA (<10⁸ capsids/ml, see above).

We could confirm that VLPs can be generated from expression constructscomprising some sequence 5′ of the VP3 start codon together with thesequence coding for VP3, but in contrast to the results of Hoque et al.,we could not quantify capsid assembly in detectable amounts (>10⁸capsids/ml, see example 8) using the NLS_(SV40)-VP3 fusion construct.Accordingly, the method of Hoque et al. is not suitable for thegeneration of large amount of pure VLPs suitable for vaccinationpurposes for the market.

Taken together, the prior art techniques either use expression systemsin the presence of Rep inevitably leading to the packaging of Rep andDNA or in the absence of Rep yields of VP3 VLPs are too low in order togenerate a commercially viable process or product.

Accordingly, it was an object of the present invention to provideparticles useful as a vaccine based on VLPs and methods of producing thesame avoiding one or more of the above disadvantages. Particularly, itis desirable that the VLPs essentially consist of only one type of viralprotein, contain no or only very little amounts of DNA and/or that theymay be produced in an economical manner, e.g. in high yields.

The problem is solved by providing parvoviral particles consistingessentially of VP3, with essentially no VP1, VP2 and Rep proteins. Theymay be produced by expressing in a cell VP3 from a VP3 coding sequence(cds) of the parvoviral structural protein VP3 (VP3 cds) under controlof a rep-independent promoter. Additionally, in this method a DNAsequence fragment (fragment Z) (partially) encoding a newly identifiedpolypeptide designated “assembly activating protein” (AAP) is expressed,which allows for high yields, e.g. approximately about 10⁵, preferablyabout 10⁶, and more preferably about 10⁷ virus particles to be formedper cell. The identification of this novel protein is a totally newconcept with respect to the assembly of parvoviral capsids in generaland especially for VP3 capsids, as no sequence motif within a VP2protein such as the postulated “PARKRL” motif or a heterologous nuclearlocalization sequence for VP3 is required as postulated (Hoque et al.,1999a, 1999b).

In contrast to the state of the art these VLPs do not contain aheterologous NLS or a VP2 protein. Upon epitope insertion at one orseveral of the preferred sites in the VP3, particles could besuccessfully assembled that presented epitopes for vaccine development.With this method 10¹¹, preferably about 10¹², and more preferably about10¹³ virus particles are formed per ml crude lysate and therefore yieldsare sufficient for a commercially viable product.

Surprisingly and in line with its function of encoding a polypeptide,the sequence fragment Z can be provided either in cis or in trans toassemble capsids consisting essentially of VP3. Further, fragment Z andVP3 can be derived from the same or different species of parvovirusfamilies, mutually trans-complementing each other regarding VP3 particleassembly.

The following definitions explain how the defined terms are to beinterpreted in the context of the products, methods and uses of thepresent invention.

“AA” is used as abbreviation for amino acid(s), “nt” is used asabbreviation for nucleotide(s).

According to this invention a “parvovirus” or “parvoviral” relates to amember of the family of Parvoviridae wherein the wildtype expresses VP1,VP2 and VP3 as capsid proteins. The family of Parvoviridae containsseveral genera divided between 2 subfamilies Parvovirinae (Parvovirus,Erythrovirus, Dependovirus, Amdovirus and Bocavirus) and Densovirinae(Densovirus, Iteravirus, Brevidensovirus, Pefudensovirus andContravirus) (Fields: Virology, fourth edition 2001, Volume 2, chapters69 and 70, Lippincott Williams Wilkins, Philadelphia;http://virus.stanford.edu/parvo/parvovirus. htmlhttp://www.ncbi.nlm.nih.gov/ICTVdb/lctv/fs_parvo.htm#SubFamily1). Thewildtype capsid is assembled of the three structural proteins VP1, VP2and VP3 that form the 60 subunits of the AAV capsid in a ratio of 1:1:8(Kronenberg et al., 2001). Hence, the term “VP3” stands for virusprotein 3. The naturally occurring parvoviral particle is composed ofthe icosahedral capsid that encloses the single stranded DNA genome.Preferred parvoviruses are the Dependoviruses, including AAV.

In the context of this invention the term “serotype” stands for the kindof virus of a group of closely related viruses distinguished by theircharacteristic set of antigens. Thus, the serotype is characterized byserologic typing (testing for recognizable antigens on the virussurface). Accordingly, the AAV can also be selected from a serotypeevolved from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9,AAV10, AAV11 to AAV12 and AAV13, in particular from AAV2.

Parvoviral particles consisting “essentially of VP3” or “essentiallyonly VP3” means that the capsid is assembled to at least 98%, preferablyat least 99%, more preferably at least 99.6% and essentially at least99.8% of VP3. This means that only 1/50, preferably 1/100, morepreferably 1/250 and essentially only 1/500 or less of the proteinsassembling the capsid are N-terminally extended versions of VP3 orcompletely different proteins. In a preferred embodiment the capsid isassembled to at least 98%, preferably at least 99%, more preferably atleast 99.6% and essentially at least 99.8% of VP3, meaning that only1/50, preferably 1/100, more preferably 1/250 and essentially only 1/500or less of the proteins assembling the capsid are N-terminally extendedversions of VP3 or different parvoviral proteins. It is especiallypreferred that the parvoviral capsid consists only of one protein, whichis VP3 in its wildtype sequence or a mutated form of it.

A “coding sequence” or “cds” means that portion of a gene which directlyspecifies the amino acid (AA) sequence of its product. Hence, the “VP3coding sequence” or “VP3 cds” defines that part of the cap gene fromwhich the genetic code is translated into the amino acid (AA) sequenceof a VP3, which can be wildtype or mutated as further defined in thisinvention. The VP3 cds is located at the 3′ end of the cap ORF andstarts with an ATG nucleotide triplet coding for a methionine. Dependingfrom the individual parvovirus chosen, the VP3 cds is translated intoabout 533 Aas. E.g. for AAV2 the cds of the major coat protein VP3 canbe obtained from the NCBI entree (http://www.ncbi.nlm.nih.gov/)NC_001401 (nucleotides 2809-4410) according to Ruffing et al. (1994),the AA sequence from the corresponding NCBI entree YP_680428. A VP3 cdsaccording to this invention encodes a VP3 protein which is capable ofparticle formation according to the methods of this invention. AnN-terminally extended VP3 protein comprises one or more of therespective Aas of VP2. Accordingly, VP2 can be seen as an N-terminallyextended VP3, in contrast to a VP3 which has an N-terminal insertion ofa heterologous sequence thereto, such as a Tag or an epitope as furtherdefined below.

The genetic code defines a mapping between tri-nucleotide sequences,called “codons”, and Aas. A triplet codon in a nucleic acid sequenceusually specifies a single AA.

A “reading frame” is a contiguous and non-overlapping set oftri-nucleotide codons in DNA or RNA. There are 3 possible reading framesin an mRNA strand and six in a double stranded DNA molecule due to thetwo strands from which transcription is possible. An “open readingframe” (ORF) is a reading frame that contains a start codon, thesubsequent region which usually has a length which is a multiple of 3nucleotides, and ends with a stop codon. An ORF could potentially encodea protein. Insertion of one or two nucleotides unambiguously results ina shift to a different reading frame (frameshift mutation). Usually, ATGis used as the start codon. However, as already known from VP2 of AAVnon-canonical start codons are sometimes used.

“Mutations” are changes to the nucleotide sequence of the geneticmaterial of an organism. Such mutations may lead to a change of theencoded protein and therefore may have varying effects depending onwhere they occur and whether they alter the structure and/or function ofthe encoded protein. Structurally, mutations can be classified as pointmutations, insertions adding one or more extra nt into the DNA/AA intothe protein or deletions removing one or more nt/AA. An “insertion” ofnt/AA is generally speaking an insertion of at least one heterologousnt/AA into the sequence of—for this invention—a parvovirus protein.‘Heterologous’ in this context means heterologous as compared to thevirus, from which the parvovirus protein is derived. Exemplified for aparvovirus structural protein, the inserted Aas can simply be insertedbetween two given Aas of the parvovirus structural protein. An insertionof Aas can also go along with a deletion of given Aas of the parvovirusstructural protein at the site of insertion, leading to a completesubstitution (e.g. 10 given Aas are substituted by 10 or more insertedAas) or partial substitution (e.g. 10 given Aas are substituted by 8inserted Aas) of Aas of the parvovirus structural protein.

In addition to an open reading frame beginning with a start codon closeto its 5′ end some further sequence requirements in the localenvironment of the start codon have to be fulfilled to initiate proteinsynthesis. One of these is the “Kozak sequence”. The amount of proteinsynthesized from a given mRNA is dependent on the strength of the Kozaksequence. For a ‘strong’ consensus, relative to the translationinitiation codon that is referred to as number 1 the nucleotides atpositions +4 (i.e. G in the consensus) and -3 (i.e. either A or G in theconsensus) must both match the consensus (there is no number 0position). An ‘adequate’ consensus has only 1 of these sites, while a‘weak’ consensus has neither. The cc at −1 and −2 are not as conserved,but contribute to the overall strength. There is also evidence that a Gin the −6 position is important in the initiation of translation.

The term “percent identity” with respect to two sequences, particularamino acid sequences, indicates how many amino acids or bases areidentical in an alignment of two sequences. For normalization, eitherthe length of longer sequence, of shorter sequence or of columns ofalignment occupied in both sequences, may be used. Usually, sequencealignment software is used in order to determine percent identity ofsequences. Common software tools used for general sequence alignmenttasks include for example ClustalW and T-coffee for alignment, and BLASTand FASTA3x for database searching. The skilled person will be able toselect a suitable method or software and appropriate settings whenassessing percent identity.

“Nucleic acid molecule” may be in the form of RNA, such as mRNA or cRNA,or in the form of DNA, including, for instance, cDNA and genomic DNAe.g. obtained by cloning or produced by chemical synthetic techniques orby a combination thereof. The DNA may be triple-stranded,double-stranded or single-stranded. Single-stranded DNA may be thecoding strand, also known as the sense strand, or it may be thenon-coding strand, also referred to as the anti-sense strand.

A “Rep-independent promoter” is a promoter which can be activated in theabsence of the Rep protein, whereas in the context of this invention Repstands for the non-structural protein(s) encoded by a parvovirus,particularly Rep40, Rep52, Rep68 and Rep78 as described by Muzyczka andBerns (2001). These promoters include for example heterologousconstitutive promoters and inducible promoters.

“Gene expression” is the process by which inheritable information from agene, such as the DNA sequence, is made into a functional gene product,such as protein or nucleic acid. Thus, gene expression always includestranscription, but not necessarily translation into protein. rRNA andtRNA genes are an example for non-protein coding genes that areexpressed into rRNA and tRNA, respectively, and not translated intoprotein. In order for gene expression to take place a promoterpreferably has to be present near the gene to provide (a) bindingsite(s) and recruit (an) enzyme(s) to start transcription.

“Shut off” of gene expression means that expression of a gene isblocked. It may be either through genetic modification (a change in theDNA sequence including mutation or deletion of the start codon, at leastpart of the cds or at least part of a sequence element necessary for itsexpression like e.g. the promoter), or by treatment with a reagent suchas a short DNA or RNA oligonucleotide with a sequence complementary toeither an mRNA transcript or a gene. Latter can preferably be used fortransient shut off.

“Poly (A)” sites at the 3′ end of the transcript signal the addition ofa series of adenines during the RNA processing step before migration tothe cytoplasm. These so-called poly(A) tails increase RNA stability.

The “sequence fragment Z” or “fragment Z” is a DNA fragment thatcomprises

(i) at least 44 nucleotides upstream of the VP3 start codon and(ii) more than 242 nucleotides of the VP3 cds starting with the startcodon,derived from

-   (a) a parvovirus, or-   (b) a nucleotide sequence that is at least 60%, preferably 80%, more    preferably 90%, especially 99% and advantageously 100% identical to    the nucleotide sequence of fragment Z derived from AAV2 (sequence 1,    FIGS. 2A-2D), or-   (c) a nucleic acid sequence that hybridizes in 4×SSC, 0.1% SDS at    65° C. to the complementary strand of the fragment Z DNA molecule of    AAV2, or-   (d) a nucleic acid sequence that can be used in    trans-complementation assays to cause assembly of VP3 VLPs.

This means that the sequence of fragment Z at the same time representspart of the VP2 and VP3 cds, since the AAV capsid genes are encoded byoverlapping sequences of the same ORF using alternative mRNA splicingand alternative translational start codons. Thus, the VP2 gene containsthe whole VP3 gene sequence with a specific 5′ region (schematicrepresentation in FIG. 1).

A “functionally active variant” of the claimed polypeptide or a nucleicacid is a polypeptide or a nucleic acid that is referred to in thecontext of the present invention is a variant obtained by one or moremutations as detailed herein, which is functionally active in that thevariant maintains its biological function, e.g. its capability topromote assembly of VP3. The biological activity may be determined intrans-complementation assays, where the expression of such polypeptidefrom such nucleic acid is able to promote assembly of VP3 VLPs from aVP3 coding construct whose expression under suitable conditions isinsufficient for VP3 capsid assembly. Suitable insufficient AAV2 VP3coding constructs are pCMV-VP3/2809 or pCI-VP3. A suitable test isdescribed in the Examples, e.g. in Example 3. Preferably, maintenance ofbiological function is defined as having at least 50%, preferably atleast 60%, more preferably at least 70%, 80% or 90%, still morepreferably 95% of the activity of the natural occurring AAP.

Complementation assays can be performed as described in example 3 andeither be analyzed by ELISA (example 1.5) or by immunofluorescence(1.6). Both assays are based on the detection of virus particles by thebinding of a monoclonal antibody to the viral capsid in an assembledstate. For example the monoclonal antibody A20 (Progen, Heidelberg,Germany) binds to the viral capsid of AAV2 and some other AAV serotypes,for more distantly related serotypes specific antibodies arecommercially available. If no specific antibody is available, viralcapsids can be detected by electron microscopy (for example see Hoque etal. (1999b)), or sucrose density gradient analysis (example 1.3.2.)

“Extended versions of VP3” comprise in general N-terminal extensions byseveral Aas. These N-terminal extensions represent the 3′ part of thesequence coding for VP2 but not for VP3, since the AAV capsid genes areencoded by overlapping sequences of the same ORF using different startcodons (FIG. 1). Thus, N-terminally extended VP3 is identical toN-terminally truncated VP2 meaning that parts of VP2 can be presentwithin the N-terminal extension of VP3 but no complete and intactwildtype VP2 protein is expressed as e.g. given by Ruffing et al. (1994)and accessible from NCBI (number of entree: NC_001401. According to thisinvention the particles consist essentially of VP3 (as defined) andtherefore extended versions of VP3 are very rare, whereas naturallyoccurring particles comprise VP1:VP2:VP3 in a ratio of 1:1:8 (Kronenberget al., 2001).

To determine the composition of capsid proteins expressed in a givensample Western blot analysis can be used. The cell lysate or purifiedVLPs can be fractionated on a sucrose gradient and fractions analyzedupon gel electrophoresis and transfer to a nitrocellulose membrane,where they can be probed using binders specific to the target protein.The monoclonal antibody B1 reacts with all three capsid proteins and canbe used to detect VP3, whereas the monoclonal antibody A69 reacts onlywith VP1 and VP2 and can be used to detect truncated VP2.

In the context of this invention “efficient particle formation” meansthat a high titer of particles is formed of about 10¹¹, preferably ofabout 10¹², and more preferably of about 10¹³ particles/ml in crudelysate (corresponding to about 10⁵, preferably about 10⁶, and morepreferably about 10⁷ particles/transfected cell).

The term “about” means according to the invention a general error rangeof ±20%, especially ±10%, in particular ±5%.

Virus particle titers can be quantified from lysates of transfectedcells (see above) in their undiluted form or in a dilution using acommercially available titration ELISA kit which is based on the bindingof the monoclonal antibody A20 to the viral capsid in an assembled stateto measure the virus concentration. As already described above, if theantibody A20 does not bind to the capsid of e.g. a different virusserotype, particle titers can be visualized by electron microscopy andquantified by counting (Grimm et al., 1999, Grimm and Kleinschmidt,1999, Mittereder et al., 1996).

To analyze protein expression and estimate its amount cell lysates ofidentical portions of transfected cells can be processed for SDS-PAGE.Upon gel electrophoresis and transfer to a nitrocellulose membrane,proteins can be probed using binders specific to the target protein(e.g. monoclonal antibodies B1, A69, anti-GFP). The amount of proteintranslation can be estimated from the amount of binders thatspecifically bind to the protein. These complexes can be visualized andquantified by e.g. immunohistochemical staining, immunofluorescentstaining or radioactive labeling.

The term “binder” refers to a molecule that specifically binds to itsrespective binding partner. Commonly used binders are antibodies,especially monoclonal antibodies, antibody derivatives such as singlechain antibodies or antibody fragments. In principle all classes ofantibodies can be used, preferred are IgG antibodies. Fragments ormultimers of antibodies can equally be used. Commonly used fragments aresingle chain antibodies, Fab- or (Fab)₂-fragments. Examples of othersuitable binders are protein scaffolds such as anticalins or lipocalins(Nygren and Skerra, 2004), receptors or parts thereof (e.g. solubleT-cell receptors), ankyrine, microbodies or aptamers.

The term “specifically binds” means that two molecules A and B,preferably proteins, bind to each other thereby generating complex ABwith an affinity (K_(D)=k_(off)/k_(on)) of at least K_(D)=1×10⁻⁵ mol/l,preferably at least 1×10⁻⁷ mol/l, more preferably at least 1×10⁻⁸ mol/l,especially at least 1×10⁻⁹ mol/l.

An “epitope” is the part of a macromolecule that is recognized by theimmune system, specifically by antibodies, B-cells, or T-cells.

A “mimotope” is a non-linear structural epitope composed of several Aasderived from different regions of the linear sequence of the structuralprotein located in close neighborhood due to the overall tertiarystructure of the capsid or from a non-peptide structure such ascarbohydrate residues, nucleic acids or lipids, and such non-linearstructural epitope is specifically bound by an antibody. Thus, bymimicking the structure of an epitope the mimotope causes an antibodyresponse identical to the one elicited by the epitope. The mimotope inthe context of the present invention might consist of (parts of) theinserted peptide sequence alone or might be composed of inserted peptideand parvovirus core particle AA residues.

As used herein the term “B-cell epitope” is meant to include alsomimotopes. Therefore, the epitopes can be both linear and structural.

The term “antigen” in the context of the products, methods and uses ofthe present invention refers to any target antigen against which animmune reaction should be induced. Such target antigens are usuallyantigens that are susceptible to the humoral immune response. They areusually proteins that may be posttranslationally modified, as forexample glycosylated proteins.

The term “Immunoglobulin” (abbr. Ig) refers to any of the glycoproteinsnaturally occurring in the blood serum that are induced in response toinvasion by immunogenic antigens and that protect the host byeradicating pathogens. In total, there are five human antibody classes,known as IgM, IgG, IgA, IgD and IgE, which belong to this group ofproteins.

In a first aspect, this invention relates to a nucleic acid encoding apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ IDNO: 19, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, or encoding apolypeptide comprising a functionally active variant of any of theseamino acid sequences, wherein the functionally active variant

-   (i) has an amino acid sequence that is at least 60% identical to any    of the amino acid sequences of SEQ ID NO: 1 to 22, and/or-   (ii) is encoded by a cDNA that hybridizes in 6×SSC, 5×Denhardt's    solution, 0.5% SDS at 40° C. for 2 to 12 hours to a nucleic acid    sequence selected from the group consisting of SEQ ID NO: 23, SEQ ID    NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28,    SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID    NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37,    SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID    NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44, or to a nucleic acid    sequence complementary to any of the nucleic acid sequences of SEQ    ID NO: 23 to SEQ ID NO: 44; and/or-   (iii) is encoded by a part of a parvoviral genome comprising an open    reading frame (ORF) not in frame with that encoding VP1, VP2 and    VP3, that includes more than 378 nucleotides of the VP3 ORF,    wherein the nucleic acid is incapable of expressing any of the    functional Rep proteins, particularly incapable of expressing Rep40,    Rep52, Rep68, Rep78, VP1, VP2 and VP3.

It was demonstrated that co-expression of a so far unidentified productof the AAV2 cap gene efficiently promotes assembly of VP3 into anicosahedral capsid. This protein, designated assembly activating proteinor AAP is encoded by ORF2 of the cap gene (wherein the first ORF encodesVP1, VP2 and VP3) and has a molecular weight of approximately 23 kDa.The molecular weight of AAP estimated from Western blots was higher(about 30 kDa) maybe due to posttranslational modification(s). Itscellular localization is in the nucleolus and it targets the VP proteinsto the nucleolus where capsid assembly takes place. However, nucleolarlocalization of VP3 alone is not sufficient for capsid formation,indicating that AAP provides an additional chaperon-type, scaffoldand/or nucleation function also within the full length AAV genome.

Homologous polypeptides can be identified for different parvoviruses.Such an alignment of predicted AAP protein sequences derived from ORF2of the cap gene of different parvoviruses are shown in FIG. 28.Accordingly, the nucleic acid according to the invention is preferablycharacterized in that it encodes a polypeptide comprising the amino acidsequence of SEQ ID NO: 1 (AAV2), SEQ ID NO: 2 (AAV1), or the amino acidsequence of SEQ ID NO: 5 (AAV5).

It is envisaged by this invention that naturally occurring AAP may bemodified but remains functionally active. Such functionally activevariants may be generated e.g. in order to increase expression,stability and/or activity, or in order to facilitate easier cloning ofconstructs. Accordingly, the invention also refers to an functionallyactive variant that has an amino acid sequence that is at least 65%,more preferably at least 70%, more preferably at least 75%, morepreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, more preferably at least 95%, most preferably at least 99%and especially 100% identical to any of the amino acid sequences of SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ IDNO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20,SEQ ID NO: 21, and SEQ ID NO: 22 and/or that is encoded by a cDNA thathybridizes in 6×SSC, 5×Denhardt's solution, 0.5% SDS at 45° C., morepreferably at 50° C., more preferably at 55° C., more preferably at 60°C., especially at 65° C. and advantageously at 68° C. to a nucleic acidsequence complementary to any of the nucleic acid sequences of SEQ IDNO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32,SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO:37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ IDNO: 42, SEQ ID NO: 43, and SEQ ID NO: 44. Preferably the functionallyactive variant is encoded by a cDNA that hybridizes at the conditionsspecified above in 6×SSC, 5×Denhardt's solution, 0.5% SDS to the nucleicacid sequence of SEQ ID NO: 23, or a nucleic acid sequence complementaryto the nucleic acid sequence of SEQ ID NO: 23.

In a preferred embodiment of the invention, the nucleic acid encodes apolypeptide consisting of an amino acid sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ IDNO: 19, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, or encodes apolypeptide consisting of a functionally active variant of any of theseamino acid sequences, wherein the functionally active variant is definedabove. More preferably the nucleic acid encodes a polypeptide consistingof an amino acid sequence selected from the group consisting of SEQ IDNO: 1, to SEQ ID NO: 22.

Due to N- and C-terminal truncation experiments with AAP it has beenfound that with respect to the 3′-end of AAP of AAV2 378 nt overlappingwith the VP3 ORF starting at ATG₂₈₀₉ are not able to support VP3 capsidassembly, whereas 445 nucleotides of the VP3 ORF are about equallyefficient in yield of capsids as wt AAV. Accordingly, the nucleic acidof the invention is characterized in that it includes more than 378nucleotides (such as more than 378, 379, 380, 381, 382, 383, 384, 385,386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399nucleotides), preferable at least 400 nucleotides (such as at least 400,401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414,415, 416, 417, 418, 419, 420, 421, 422, 423, nucleotides), morepreferably at least 425 nucleotides (such as at least 425, 426, 427,428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441,442, 443, 444 or 445 nucleotides), and especially at least 445nucleotides (such as 445, 446, 447, 448, 449, 450, 451, 452, 453, 454,455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468,469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482,483, 484, 485, 486, 487 or 488, 489, 490, 491, 492, 493, 494, 495, 496,497, 498, 499, 500 or more nucleotides) of the VP3 ORF.

With respect to the 5′-end of AAP of AAV2 an N-terminally truncated AAPencoded by a nucleic acid with a 44 nucleotide extension upstream of theVP3 start codon is about equally efficient in yield of capsids as wtAAV, if translation is started by an ATG inserted in frame to ORF2, andwith lower efficiency if no ATG start codon is inserted (data notshown). An N-terminally truncated AAP encoded by a nucleic acid startingwith an ATG instead of the ACG at position 2858 did not lead todetectable capsid formation. For AAV4 and AAV9 it was shown that a VP3cds expression construct starting at the respective VP3 start codon issufficient for detectable capsid assembly, therefore still encodingfunctional AAP (variant) (data not shown).

Accordingly, the nucleic acid of the invention is characterized in thatit includes at least 44 nucleotides (such as 44, 45, 46, 47, 48, 49, or50 nucleotides), preferably at least 20 nucleotides (such as 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42 or 43 nucleotides), more preferably at least 5 nucleotides (suchas 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 nucleotides)of the adjacent VP2-encoding nucleotides, which are located in directsuccession of the 5′ of the VP3 start codon.

The nucleic acid encoding AAP or variants thereof may even start 3′ ofthe VP3 start codon, as can be seen from AAV4 and AAV9 (above).Therefore, in another preferred embodiment, the nucleic acid of theinvention is characterized in that its start codon is an ATG at 4nucleotides, preferably 24 nucleotides, and more preferably 44nucleotides downstream of the VP3 start codon.

Therefore, in preferred embodiment the nucleic acid of the inventioncomprises nucleotides starting at least at 44 nucleotides upstream and445 nucleotides downstream of the VP3 start codon (counting includes theATG), preferably at least 20 nucleotides (such as 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43 or 44 nucleotides) upstream and 425 nucleotides (such as 425, 426,427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440,441, 442, 443, 444 or 445 nucleotides) downstream of the VP3 startcodon, and especially at least 5 nucleotides (such as 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18 or 19 nucleotides) upstream and 400nucleotides (such as 400, 401, 402, 403, 404, 405, 406, 407, 408, 409,410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423 or424 nucleotides) downstream of the VP3 start codon. Accordingly, totallength of the nucleic acid of the invention is at least 489 nt (such as489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500 or more nt),preferably at least 445 nt (such as 445, 446, 447, 448, 449, 450, 451,452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465,466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479,480, 481, 482, 483, 484, 485, 486, 487 or nt), and especially at least405 nt (such as 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415,416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429,430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443 or444 nt).

The nucleic acid of the invention is capable of expressing a proteinpromoting capsid assembly of VP3. It may be characterized in that it isderived from AAV2 and its translation start codon (found in wildtypeAAV2 sequences) is C₂₇₂₉TG, A₂₇₃₅CG, A₂₇₁₇TT or T₂₇₂₀TG or that it isderived from another parvovirus and its translation start codon is atthe homologous site to the translation start codons of AAV2. Homologousstart codons for other parvoviruses can easily be identified by thegiven alignment (see FIGS. 27A and 27B) and looking for amino acidsencoded by potential non-canonical start codons. Such potentialnon-canonical start codons can easily be verified by mutational analysisas done for AAV2 C₂₇₂₉TG in example 14. For parvoviruses not shown inFIGS. 27A and 27B such a sequence can easily be added to the givenalignment.

In a preferred embodiment the AAP encoding ORF is mutated in a way inorder to generate an ATG start codon allowing for improved translationof the open reading frame, whereas “improved” means higher expression ofAAP or variants thereof compared to the respective wildtype sequence.Preferably one of the translation start codons of AAV2 or the homologoussites of other parvoviruses is mutated into an ATG start codon. Startingtranslation with the canonical start codon ATG generally leads tooptimized expression of AAP or variants thereof and therefore, when AAPor variants thereof is suboptimal, leads to increased yield of capsidassembly. This becomes especially beneficial if expression systems areswitched to cells that the respective virus is not adapted to. It can beassumed that expression of AAP or variants thereof in non-host cellswill be suboptimal. For example, it is foreseen within this invention tomanufacture capsids in insect cells or other cells suitable forinfection by Baculovirus, in yeasts or bacteria, where optimizedexpression of AAP or variants thereof may be highly beneficial orcrucial in order to get high capsid formation.

Whereas such mutation of the start codon of AAP into an ATG may reducecapsid formation in a cis situation (where AAP is encoded by anoverlapping nucleic acid with ORF1 encoding VP3), such mutation isespecially beneficial in a trans situation, where AAP is encodedindependently from ORF1 encoding VP3 (example 14).

It is well known in the art and part of the invention that the nucleicacid is characterized in that the polypeptide coding sequence of thenucleic acid is followed by a poly(A) signal.

In one aspect of the invention the nucleic acid of the inventioncomprises a promoter driving transcription of the polypeptide-encodingsequence. In a preferred embodiment, a heterologous promoter, i.e. whichis not present in the virus from which AAP-encoding nucleic acid isderived or preferably not present in any parvovirus wildtype genome, isused. The promoter which can be used in the method described herein isnot limited to the examples described herein. It may be any known orsubsequently discovered one. Constitutive promoters like e. g. the earlycytomegalovirus (CMV) promoter (U.S. Pat. No. 4,168,062), that arecontinuously transcribed, are as useful in the invention as induciblepromoters such as an antibiotic-specific or a cell-specific promoter.For expression in mammalian cell systems use of the CMV promoter isespecially preferred, e.g. for use in manufacturing processes usingtransfection methods, whereas in insect cells use of the Polyhedrinpromoter (PolH) is preferred. Inducible heterologous promoters areespecially preferred, as they can be used to establish stable productioncells for VP3.

Due to the high conservation of genome organization amongst theparvoviruses, the invention can easily be transferred to otherparvovirus members. Within the parvoviruses preferred viruses, fromwhich the nucleic acid of the invention is derived from, areadeno-associated virus (AAV), Goose parvovirus, Duck parvovirus, andSnake parvovirus. Preferred AAVs are selected from the group consistingof bovine AAV (b-AAV), canine AAV (CAAV), mouse AAV1, caprine AAV, ratAAV, avian AAV (AAAV), AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8,AAV9, AAV10, AAV11, AAV12, and AAV13, especially AAV2.

In a further aspect the nucleic acid of the invention is comprised in anexpression cassette, construct, vector or cell line. A construct,typically a plasmid, is generally a nucleic acid comprising the nucleicacid of the invention and additional sequences such as polycloningsites, origin of replication, selection marker genes etc. An expressioncassette is generally a construct that, once it is inside a cell, isable to produce the protein encoded by the nucleic acid of the inventionby the cellular transcription and translation machinery. The expressionconstruct is engineered to contain regulatory sequences that act asenhancer or promoter regions and lead to efficient transcription of thenucleic acid of the invention. It further usually comprises apoly(A)-site that is later polyadenylated which is important for nuclearexport, translation and stabilization of the mRNA. Vectors areconstructs that are used to introduce the nucleic acid of the inventioninto cells. Dependent on the cells to be transfected they areconstructed according to standard skills of the artisan. These can beplasmids for calcium phosphate transfection or liposomal transfection,or viral vectors, e.g. baculoviruses. Cell lines are laboratory celllines suitable for the expression of AAP or variants thereof or thereplication of AAP (variant) encoding plasmids.

A further aspect of the invention is a polypeptide encoded by a nucleicacid according to the invention. The underlying naturally occurringpolypeptide is referred to as Assembly Activating Protein (AAP).Accordingly, variants of this polypeptide encoded by the nucleic acid ofthe present invention are referred to as APP variants. For example, avariant comprising the AAP protein and one or more further peptideswould be referred to as an AAP-comprising polypeptide. The protein AAPis expressed from ORF2 (with the start codon for VP3 defining ORF1), hasa calculated molecular weight of approximately 23 kDa and is able toprovide capsid assembly of VP3 in the nucleolus. It is also essentialfor capsid formation within the whole AAV genome. It targets VP proteinsto the nucleolus and exerts there an additional function in promotingthe assembly reaction.

A further aspect of the invention is a method of producing thepolypeptide of the invention, i.e. AAP or an AAP variant, by expressinga nucleic acid according to this invention in a host cell. Suchproduction is suitable to promote capsid formation of parvoviruses ingeneral and specifically of capsid comprising VP3, but no VP1 and VP2and Rep proteins. Suitable host cells can be selected by the skilledperson according to his needs and preferences. Preferred host cellsselected from a list consisting of a mammalian cell line, especially ahuman cell line, a cell line used for baculovirus infection, a bacterialstrain and a yeast strain.

A further aspect of the invention is an antibody or a binder in generalthat specifically binds AAP. Particularly, the antibody specificallybinds to any of the sequences of SEQ ID NO: 1 to 22. Such antibodies canbe used to further investigate the function of AAP or, when used as atransacting factor in heterologous expression systems, in order toverify and optimize AAP expression levels for commercial production ofparvoviruses DNA or virus like particles. A preferred antibody ischaracterized in that it specifically binds AAP of AAV2 (SEQ ID NO:1).Antibodies according to this invention may be polyclonal or monoclonal.Further encompassed by the invention are corresponding antibodyfragments like single chain antibodies, scF_(v)s, F_(ab) fragments,nanobodies or alike, or antibody multimers.

A further aspect of the invention is the use of nucleic acid of theinvention for the preparation of a polypeptide of the invention,including AAP and AAP variants.

A further aspect of the invention is the use of the nucleic acid or thepolypeptide of the invention for the preparation of a parvovirus andparvoviral particle. The identification of AAP leads to previouslyunknown possibilities to manufacture such viruses as expressionconstructs can be optimized individually in order to increase yield orin order to generate inducible production systems using stabletransfected producer cell lines. Expression can be increased through theuse of heterologous promoters. Specifically, particles can be preparedin the absence of functional Rep and VP1 and VP2 encoding sequencesenabling the manufacture of parvoviral particles not comprising any ofthe functional proteins VP1, VP2, Rep40, Rep52, Rep68 and Rep78. Allthese factors are important in the context of generating a robust, fastand cheap production system for such viruses and particles.

One aspect of the invention is a method of producing parvoviralparticles consisting essentially of VP3, the method comprising the stepsof (i) providing a cell capable of expressing VP3 from a VP3-codingsequence (cds) from a parvovirus, wherein the VP3 is under control of arep-independent promoter and expressing a protein encoded by the nucleicacid according to the invention, (ii) incubating the cell at conditionsconducive to the expression of VP3 and the protein from the nucleic acidaccording to the invention, thereby producing the parvoviral particle,and (iii) optionally purifying parvoviral particles from the cell,wherein at least 10⁵ virus particles are formed per cell and nofunctional VP1, VP2, Rep40, Rep52, Rep68 and Rep78 proteins areexpressed. This method is equally applicable using fragment Z instead ofthe nucleic acid according to the invention.

In another aspect the invention provides a method of producingparvoviral particles essentially consisting of VP3, comprising the stepsof

-   i. expressing VP3 from a VP3 coding sequence (cds) from a parvovirus    under control of a rep-independent promoter in a cell,-   ii. expressing a DNA sequence fragment (fragment Z) in the cell    under control of a rep-independent promoter, that comprises    -   (1) at least 44 nucleotides upstream of the VP3 start codon and    -   (2) more than 242 nucleotides of the VP3 cds starting with the        start codon    -   derived from    -   a) a parvovirus, or    -   b) a nucleotide sequence that is at least 60%, preferably 80%,        more preferably 90%, especially 99% and advantageously 100%        identical to the nucleotide sequence of fragment Z derived from        AAV2 (sequence 1, FIGS. 2A-2D), or    -   c) a nucleic acid sequence that hybridizes in 4×SSC, 0.1% SDS at        65° C. to the complementary strand of the fragment Z DNA        molecule of AAV2 (sequence 2), or    -   d) a nucleic acid sequence that can be used in        trans-complementation assays to cause assembly of VP3 VLPs.-   iii. incubating the cell at conditions suitable for VP3 expression,    and-   iv. purifying parvoviral particles from the cell,    wherein approximately about 10⁵, preferably about 10⁶, and more    preferably about 10⁷ virus particles are formed per cell and    essentially no VP1, VP2 and Rep proteins (particularly Rep40, Rep52,    Rep68 and Rep78) are expressed.

The invention of these methods is based on the generation of particlesfrom a virus of the family of Parvoviridae wherein the wildtypeexpresses VP1, VP2 and VP3 as capsid proteins. Parvoviral particlesconsisting essentially of VP3 may be generated by expressing theparvoviral VP3 cds essentially in the absence of expression offunctional VP1, VP2 and Rep proteins, particularly Rep40, Rep52, Rep68and Rep78. As a result, the purified parvoviral particle consistsessentially of only one capsid protein. Rep-mediated DNA packaging iscompletely avoided due to the absence of Rep in the particle. Theinvention provides high titers of parvoviral particles consistingessentially of VP3 which are amongst others suitable for vaccinedevelopment.

It is well known in the art that VP3 alone is not able to assemble intocapsids. In the context of this invention a nucleic acid encoding anovel polypeptide designated AAP respectively a sequence element Z(fragment Z) was identified that, if expressed in the cell, mediatesassembly of VP3 particles and that VP3 does not need additional viralproteins for capsid assembly.

Several lines of evidence led to the conclusion that VP3 requires RNAderived from the cap gene for capsid assembly. This factor required forVP3 capsid assembly could be provided in trans in a fragment of the capgene fused to gfp (VP2N-gfp). Protein expression from the first ORF ofthis cap gene fragment (ORF that encodes VP1, VP2 and VP3) was notnecessary as several constructs containing stop codons in the relevantregion of the cap gene also provided helper function. Expression ofVP2N-gfp from read-through transcripts could not be detected by Westernblot analysis. Such protein expression, initiated at non-conventionaltranslation start sites and followed by a stop codon is very unlikelyand their amount would be very low. Such protein expression of VP2N-gfpis also not sufficient for stimulating capsid assembly of VP3. This hasclearly been shown by expression of this protein using alternativecodons which resulted in high VP2N-gfp protein levels but not in VP3capsid assembly. Because such a change of the codons implicates a changeof the nucleotide sequence it is clear that the correct nucleotidesequence is necessary for the assembly helper effect and not theexpressed protein of the first ORF. Finally, providing the correctnucleotide sequence by a plasmid which could not be transcribed in thefirst ORF resulted also not in capsid assembly, arguing thattranscription of the correct nucleotide sequence is necessary.

As shown in FIGS. 2A-2D, fragment Z comprises at least 44 nucleotidesupstream and more than 242 nucleotides downstream of the VP3 startcodon. Preferably, fragment Z does not comprise a full-length VP3 cds.The sequence of fragment Z can be derived from one of a number ofdifferent parvoviruses as listed in FIGS. 2A-2D where some examples forthe nucleotide sequence of the respective region for fragment Z ofparvoviruses AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10,AAV11, and b-AAV are given. This listing is not limited to theparvoviruses shown here. A further sequence can easily be alignedthrough its position of the VP3 start codon and selected as fragment Z.A nucleotide sequence can also be selected as fragment Z by its identityto the nucleotide sequence of fragment Z derived from AAV2 (SEQ ID NO:45, see below) which is at least 60%, preferably 80%, more preferably90%, especially 99% and advantageously 100%. Moreover, a nucleotidesequence hybridizing in 4×SSC, 0.1% SDS at 65° C. to the complementarystrand of the fragment Z DNA molecule of AAV2 (SEQ ID NO: 46, see below)can also be used in trans-complementation assays as fragment Z to causeassembly of VP3 VLPs. It is especially preferred that fragment Z isderived from AAV2 and comprises SEQ ID NO: 45.

Nucleotide sequence of DNA sequence fragment Z derived from AAV2 (SEQ IDNO: 45, as also given in FIGS. 2A-2D):

1 tcggacagcc accagcagcc ccctctggtc tgggaactaa tacgatggct 51 acaggcagtggcgcaccaat ggcagacaat aacgagggcg ccgacggagt 101 gggtaattcc tcgggaaattggcattgcga ttccacatgg atgggcgaca 151 gagtcatcac caccagcacc cgaacctgggccctgcccac ctacaacaac 201 cacctctaca aacaaatttc cagccaatca ggagcctcgaacgacaatca 251 ctactttggc tacagcaccc cttgggggta ttttgac

Reverse and complementary sequence of SEQ ID NO: 45, that can be used inhybridization experiments to identify an unknown DNA fragment asfragment Z (SEQ ID NO: 46):

1 gtcaaaatac ccccaagggg tgctgtagcc aaagtagtga ttgtcgttcg 51 aggctcctgattggctggaa atttgtttgt agaggtggtt gttgtaggtg 101 ggcagggccc aggttcgggtgctggtggtg atgactctgt cgcccatcca 151 tgtggaatcg caatgccaat ttcccgaggaattacccact ccgtcggcgc 201 cctcgttatt gtctgccatt ggtgcgccac tgcctgtagccatcgtatta 251 gttcccagac cagagggggc tgctggtggc tgtccga

For initiation of transcription of the VP3 cds and the sequence offragment Z or of the nucleic acid of the invention one or two“Rep-independent promoter(s)” is/are chosen. A rep-independent promoteris used in order to express VP3 and fragment Z in absence of theparvoviral factor Rep which is to be avoided as Rep is held responsiblefor packaging of virus genomes and unspecific DNA into parvoviralparticles. For the purposes of this invention packaging of viral orunspecific DNA is to be avoided as the parvoviral particles could thenunintentionally act as gene therapy vectors. By using a “Rep-independentpromoter” for VP3 expression and transcription of fragment Z or thenucleic acid of the invention, RNA polymerase can initiate transcriptionin the absence of expression of Rep proteins enabling manufacture ofcapsids in the absence of Rep proteins, particularly Rep40, Rep52, Rep68and Rep78. Rep-independent promoters are for example heterologousconstitutive or inducible promoters.

Accordingly in one aspect of the invention the nucleic acid of theinvention comprises a promoter driving transcription of thepolypeptide-encoding sequence. In a preferred embodiment a heterologouspromoter, which is not present in any parvovirus wildtype genome, isused. The promoter which can be used in the method described herein isnot limited to the examples described herein. It may be any known orsubsequently discovered one. Constitutive promoters like e. g. the earlycytomegalovirus (CMV) promoter (U.S. Pat. No. 4,168,062), that arecontinuously transcribed, are as useful in the invention as induciblepromoters such as an antibiotic-specific or a cell-specific promoter.For expression in mammalian cell systems use of the CMV promoter isespecially preferred, e.g. for use in manufacturing processes usingtransfection methods, whereas in insect cells use of the Polyhedrinpromoter (PolH) is preferred. Inducible heterologous promoters areespecially preferred, as they can be used to establish stable productioncells for VP3.

Suitable conditions for VP expression are well known in the art and canin principle be transferred to the expression of VP3 only. To produceparvoviruses or specifically parvoviral particles the respective DNAsequences have to be transfected into cells. One protocol is describedwithin the examples. However, different transfection methods, differentcells or stably transfected cells may be used instead. Differentproduction methods are described for example by Grimm et al. (2002) andGrieger and Samulski (2005).

The methods of this invention lead to high yields of parvovirusparticles, wherein about 10⁵, preferably about 10⁶, and more preferablyabout 10⁷ virus particles are formed per transfected cell. These numberscorrespond to about 10¹¹, preferably about 10¹², and more preferablyabout 10¹³ particles/ml of crude lysate. The commercial use of VP3particles requires an efficient method of production providing highyields of particles.

The particles can be purified by methods disclosed herein and the priorart.

It is especially preferred that the sequence of fragment Z or thenucleic acid according to the invention and the VP3 cds are arranged andexpressed in such a way that parvoviral particles consisting only of VP3are produced. “Consisting only” in this context means that no otherproteinaceous molecules can be detected as part of the particles bycommon methods such as Western blotting. Such particles may compriseother molecules or salts such as water and other constituents ofbuffers. Additionally, the particle may comprise molecules that areincapsulated by chance during assembly of the particle within the cell.

According to one embodiment of the invention the sequence of fragment Zor the nucleic acid according to the invention do not overlap with theVP3 cds leading to parvoviral particles consisting only of VP3. Thisavoids the expression of a substoichiometrical number of N-terminallyextended VP3 proteins present in the particles (see example 4). Such asmall number of N-terminally extended VP3 proteins most likely would notaffect activity or yield of the particles. However, under regulatoryaspects of medicaments it is advantageous to have a one-protein product.

Accordingly, it is especially preferred that the parvoviral particlesaccording to this invention are assembled only of VP3. For this purposeexpression of VP1, VP2 and Rep, particularly Rep40, Rep52, Rep68 andRep78, is shut off in the cell by a method well known to the skilledperson, as for example deletion or mutation of the respective startcodon, deletion (in whole or in part) of the cds specific for theprotein, or mutation of the cds specific for the protein, avoidingexpression of a functional gene product (examples are described forexample in Warrington et al. (2004)).

Selection of the translational initiation site in most eukaryotic mRNAsappears to occur via a scanning mechanism which predicts that proximityto the 5′ end plays a dominant role in identifying the start codon. This“position effect” causes that the first (most upstream) ATG start codonof a transcript initiates translation (Kozak, 2002).

Referring to the expression of parvovirus/AAV capsid proteins thismeans, that the minor spliced transcript mainly accounts for thesynthesis of VP1 from the first ATG whereas translation of VP3 isprimarily initiated from its ATG start codon which is the most upstreamATG of a major spliced transcript. This major spliced RNA also encodesthe unusual ACG start codon of VP2 upstream of the VP3 start site.Therefore, in addition to VP3 that is effectively synthesized from themajor spliced transcript, to a certain extent VP2 is expressed (Becerraet al., 1988, Becerra et al., 1985).

In general, the position effect is evident also in cases where amutation inactivates or removes the normal start site and translationshifts to a downstream start site. Thus, a silent internal ATG codon canbe activated and translational efficiency is increased, a problem wellknown in some disease states (Kozak, 2002).

Taken this knowledge into account, the mutagenesis of VP1 and VP2 startcodons to inactivate their expression can activate translation oftruncated proteins starting at downstream sites that are silent in thewildtype (as described by Warrington et al. (2004), and observed inexample 2.2.).

Therefore, in addition to the main start codons known for capsidproteins such alternative start codons are preferably deleted or mutatedto ensure that VP3 is the sole capsid protein to be expressed.Expression of VP3 only and shut off of any other capsid proteins may becontrolled via Western blotting as described.

In a further preferred embodiment coding sequences for VP1 and VP2,which do not encode VP3 sequences, are completely deleted from theexpression cassette encoding VP3. In such case fragment Z or the nucleicacid of the invention is provided in trans in order to enable productionof VP3 capsids.

In a preferred embodiment of the invention the DNA sequence of fragmentZ or the nucleic acid according to the invention is followed by apoly(A) signal. The poly(A) signal is able to recruit thepolyadenylation machinery to add a stretch of adenines (the poly(A)tail) onto the RNA molecule once transcription of a gene has finished.This processing step increases stability of the factor transcribed fromfragment Z within the cell. Poly(A) signals such as the poly(A) fromSV40 large T-antigen are well known in the art and are regularly used inall kinds of expression cassettes and constructs.

Our analyses of a series of deletion mutants that started expression atdifferent sites 5′ of the VP3 start codon showed that the mutantpCMV-VP3/2765 is still able to cause capsid assembly (example 2.).Therefore, as already described above, fragment Z has to comprise atleast 44 nucleotides upstream of the VP3 start codon. Since efficiencyof particle formation was increased by using a fragment Z 5′ extended bysome nucleotides it is preferred that fragment Z comprises at least 113nucleotides or especially at least 198 nucleotides upstream of the VP3start codon, respectively. In our experiments we have chosen a constructproviding a fragment Z of AAV2 that starts at nucleotide 2696(corresponding to 113 nucleotides upstream of the VP3 start codon). InFIGS. 2A-2D the sequences of the different serotypes are listed relativeto the VP3 start codon which is underlined. The sequences easily can beextended in the 5′ or 3′ direction according to the nucleotide sequencesgiven in the respective NCBI entrees (compare legend of FIGS. 2A-2D).

If the 5′ extended sequence of fragment Z comprises the translationstart codon of VP1 and/or VP2 or any other ATG start codon in ORF1, ORF2or ORF3 they have to be inactivated by mutation or deletion to expressVP3 as sole capsid protein.

Further, fragment Z has to comprise more than 242 nucleotides downstreamof the VP3 start codon. It is preferred that fragment Z comprises morethan about 275 nucleotides, more than about 300 nucleotides, more thanabout 325 nucleotides, more than about 350 nucleotides, more than about375 nucleotides, more than about 400 nucleotides, more than about 425nucleotides, and most preferably more than about 445 nucleotides of theVP3 cds starting with the start codon. An especially preferred fragmentZ stops at about nucleotide 3254 (corresponding to about 445 nucleotidesdownstream of the VP3 start codon).

The active molecule encoded by fragment Z is most likely a diffusiblemolecule, i.e. a protein designated AAP. Based on the degeneratedgenetic code we optimized the sequence of fragment Z to get potentiallyhigher expression of a putative diffusible protein possibly encodedwithin Z in the first reading frame (ORF1) that also encodes the capsidproteins VP1, VP2 and VP3, thereby leaving the AA sequence of proteinsencoded in ORF1 unchanged but disturbing the protein sequence encoded byother ORFs by modifying the DNA sequence. This codon-optimized fragment,however, could not mediate particle formation as no virus particlescould be detected anymore (example 5, FIGS. 6A-6D). On the other hand,insertion of stop codons into ORF1 within fragment Z, leading to shutoff of protein synthesis from ORF1 but leading only to minor changes inthe DNA sequence of ORF1 and not disrupting protein synthesis from ORF2,still enabled efficient particle formation (example 6, FIGS. 8A-8F).

It is especially preferred that the mutation of fragment Z comprises atleast one stop codon for protein translation in the first or thirdreading frame. As a consequence no protein with a length of 18 AA orabove can be translated from these reading frames of fragment Z inparticular no VP2 protein or part of it can be generated. Therefore itis a main advantage that no VP2 or part of it is included in theparticles.

As a preferred embodiment of this invention the main translation startcodon ATG (AA 203, numbering according to VP1 of AAV2, (Girod et al.,1999)) of VP3 within fragment Z or the nucleic acid of the invention ismutated. It is further preferred that also one or both of thealternative minor start codons of VP3 (AA 211 and AA 235, (Warrington etal., 2004)) are mutated. In a more preferred embodiment all ATG codonsthat can be used for translational start of VP3 are mutated (a number ofthe possible ones are listed in Warrington et al., 2004) to completelyavoid translation of VP3 from the expression construct providingfragment Z.

Since the product of fragment Z and the encoded function of the nucleicacid of the invention was characterized to be a trans-acting elementmeaning that fragment Z and the nucleic acid of the invention code for adiffusible molecule, the sequence of fragment Z or the nucleic acid ofthe invention can be provided on the same or a different nucleic acidmolecule to the cell as the cap gene or part thereof, e.g. a VP3 cds.

In a preferred embodiment fragment Z or the nucleic acid of theinvention is provided “in cis” relative to the VP3 cds. If fragmentZ/the nucleic acid of the invention is provided “in cis” relative to anexpression cassette coding for VP3 means that expression of fragmentZ/the nucleic acid of the invention and VP3 are driven by the same onepromoter. The sequence of fragment Z/the nucleic acid of the inventioncan be located upstream or downstream of the VP3 cds. The sequencescoding for fragment Z and VP3 can be directly linked or separated by avariable number of nucleotides (FIG. 3.1.).

In a more specific embodiment of this invention fragment Z/the nucleicacid of the invention is located directly upstream of the VP3 cds. Sincefragment Z/the nucleic acid of the invention comprises more than 242nucleotides downstream of the VP3 start codon and this sequence has notto be present in duplicate, the directly following VP3 cds has only toprovide the remaining DNA sequence of the VP3 ORF (a schema is given inFIG. 3.1.a)). In this case substoichiometrical amounts of N-terminallyextended VP3 are expressed and presented in the capsid (example 4). Inorder not to increase this part of N-terminally extended VP3 it is oneimportant embodiment of this invention not to add new or delete existingstart codon(s) respectively at the 5′ end or upstream of fragment Z/thenucleic acid of the invention.

Moreover, only the VP3-specific cds that does not overlap with fragmentZ/the nucleic acid of the invention can be mutated easily. Mutation ofthe VP3 cds that overlaps with fragment Z/the nucleic acid of theinvention also possibly changes the sequence of the diffusible moleculecoded by fragment Z/the nucleic acid of the invention. As a result, insome cases the diffusible molecule will not be active any more.Mutations in this context include silent mutations as well as e.g.insertion of epitopes. In order to increase possibilities to mutate theVP3 cds it is beneficial to minimize the overlap i.e. to separatefragment Z/the nucleic acid of the invention and the VP3 cds. This canbe done in a cis situation where one promoter drives expression of a VP3cds that does not contain the 44 nucleotides upstream of the VP3 startcodon essential for fragment Z, and of a separate fragment Z placebefore such VP3 cds or thereafter.

It is especially preferred that fragment Z/the nucleic acid of theinvention is provided “in trans” relative to the VP3 cds. If fragmentZ/the nucleic acid of the invention is provided “in trans” relative toan expression cassette coding for VP3 it means that expression offragment Z/the nucleic acid of the invention and VP3 are driven byseparate promoters (in opposite to “in cis”, see above). The sequence offragment Z/the nucleic acid of the invention can be located upstream ordownstream of the VP3 cds on the same construct, or on a differentexpression construct than the VP3 cds (examples are listed in FIG.3.2.).

In this case fragment Z comprises only the 5′ end of the VP3 cds andhence it is a main advantage that no N-terminally extended VP3 (seebelow) can be expressed and incorporated into the capsid. Therefore, ifthe sequence coding for VP3 and fragment Z are provided in trans, it isassumed that a more pure particle composition, preferably consistingonly of the structural protein VP3, can be obtained.

It is one advantage of the in trans configuration that the VP3 cds caneasily be modified e.g. to optimize its codon usage for the expressioncell line in order to further increase the yield without changing thesequence of fragment Z/the nucleic acid of the invention. Also othermodifications such as mutations, insertions, tags etc. can be donewithout affecting fragment Z/the nucleic acid of the invention. It canbe assumed that during previous attempts to identify insertion siteswithin VP3 in overlapping sequences of fragment Z and the VP3 cds,potentially useful insertion sites were not identified as an insertionalso interfered with expression of fragment Z or with the function ofAAP. Accordingly, functional separation of these sequences either in acis setting with no overlap or preferably in a trans setting enablesindependent mutagenesis of the VP3 coding sequence. Such mutagenesis hasmultiple commercial applicabilities. In the context of generation ofnovel gene therapy vectors limitations of generating chimeric parvovirusCap sequences can be overcome. Several groups have tried directevolution of AAV involving generation of randomly mutagenized virallibraries on one serotype (Koerber et al., 2008), by using STEP andshuffling methods to create multiple randomly recombined capsid speciesusing known AAV serotype capsid sequences (Ward and Walsh, 2009, Li etal., 2008). In a similar way, independent expression of AAP can be usedto identify further insertion sites that tolerate ligands (to be usedfor targeting to other cells), B-cell epitopes (to be used forgenerating epitope specific vaccines) or deletions/substitutions (to beused for detargeting of the virus or to reduce antigenicity of thevirus).

A further advantage of the in trans configuration of fragment Z/thenucleic acid of the invention and the VP3 cds is that one construct canbe stably transfected into a producer cell line whereas the otherconstruct can be transiently transfected/transduced. For example anexpression cassette comprising fragment Z/the nucleic acid of theinvention can be stably transfected into a cell line suitable forefficient VP3 expression generating a single production cell line. Suchproduction cell line then can be transiently transfected/transduced(e.g. infected with a virus) with a specific VP3 cds leading toexpression of such VP3 and respective particle formation. Accordingly,one production cell line can be used for the production of differentparticles. Given the time and cost that is needed for qualification ofproduction cell lines that are used for the manufacture of medicaments,this constitutes a considerable regulatory advantage. For this reason,it is especially preferred that fragment Z/the nucleic acid of theinvention and the VP3 cds are provided on separate expressionconstructs.

This setup can additionally be used e.g. to generate AAV/parvovirusparticles of a distinct serotype by providing an expression cassettecoding for VP3 of a specific serotype in trans to the cells that havebeen stably transfected with fragment Z/the nucleic acid of theinvention of one serotype. In general, a VP3 cds specific for an AAVserotype selected from the group consisting of AAV1, AAV2, AAV3b, AAV4,AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 and AAV13 can be usedfor transfection of cells that have been stably transfected withfragment Z/the nucleic acid of the invention of only one serotype e.g.AAV2. Thereby, AAV particles consisting of VP3 of a selected serotypee.g. AAV1 particles can easily be generated. For AAV1 and AAV2 we couldconfirm that expression of fragment Z/the nucleic acid of the inventionmediates capsid assembly of VP3 not only expressed from constructscloned from the same serotype but also expressed from constructs clonedfrom the other serotype, namely AAV2 and AAV1, respectively (example21). Nevertheless, it can not be generally expected that every serotypecomplements each other, but a person skilled in the art can easilyidentify the respective pairs of AAP from one parvovirus/serotype andVP3 from a different one to get assembly of VP3 VLPs intrans-complementation assays as described herein. Cross-complementationcan be used for example if a stable cell line expressing a specific AAPhas been obtained that can be used for the production of VP3 VLPs fromdifferent parvoviruses or AAV serotypes. The respective combinations canbe chosen for time- and cost-effective VLP production at high titer.

Just as well the other way around is possible and one specific VP3 cdscan be transfected in trans with an expression cassettes providingfragment Z/the nucleic acid of the invention chosen from a number ofdifferent sequences coding for fragment Z e.g. from different AAVserotypes/parvoviruses.

In another preferred embodiment of the invention the parvoviral particledoes not contain Rep protein, particularly any functional Rep40, Rep52,Rep68 and Rep78 proteins. For details on this embodiment are givenherein throughout the description.

In another preferred embodiment of the invention only/at most 1/50 ofthe expressed structural protein, preferably at most 1/100, morepreferably at most 1/250 and essentially only/at most 1/500 of thestructural proteins are N-terminally extended versions of VP3.Especially preferred are parvovirus particles that do not contain anystructural proteins with N-terminally extended versions of VP3 or, vicee versa, none of the structural protein are N-terminally extendedversions of VP3. If the sequences of fragment Z and VP3 cds overlap andare expressed under control of the same promoter (cis situation), theATG start codon of the VP3 cds is mainly used as start for proteintranslation whereas only a very small proportion of protein translationstarts upstream. The resulting small part of proteins contains inaddition to the VP3 cds an N-terminal extension by several Aascorresponding to the 3′ part of the sequence coding for VP2 but not forVP3. Taken together, expression of these 5′ extended versions of VP3 wasvisible in Western blots (example 4) but accounted for only 1/50 of thestructural proteins, preferably 1/100, more preferably 1/250 andessentially only 1/500 of the structural proteins of the parvovirusparticle.

In another preferred embodiment of the invention only/at most 1/50 ofthe expressed structural protein, preferably at most 1/100, morepreferably at most 1/250 and essentially only/at most 1/500 of thestructural proteins is a polypeptide according to the invention, i.e.AAP or variants thereof. Whereas no specific influence of AAP has beenshown on host cells that might have an impact on medical applications ofparvovirus vectors or particles, it is in principle beneficial to haveas little impurities as possible.

Viral Rep proteins bind to genomic and viral DNA and are discussed toplay a role in DNA packaging. In order to use AAV as a vaccine and avoidunspecific reactions against the packaged DNA or an undesired genetransfer, parvoviral particles as free of DNA as possible are especiallypreferred. Therefore, parvoviral particles are produced in the absenceof expression of Rep proteins in the cell. Hence, in still anotherpreferred embodiment of the invention only 1/100 of the particles,preferably 1/1,000 and more preferably only 1/10,000 of the particlescontain DNA. Especially preferred is that none of the parvovirusparticles contains DNA. Preferably at most 1/100, more preferablyonly/at most 1/1,000, even more preferably only/at most 1/10,000 of theparticles contain any DNA. As a result, no inactivation step to destroypackaged DNA (e.g. gamma or UV-irradiation) is necessary prior tovaccination purposes.

The parvoviruses according to this invention are preferably selectedfrom the group consisting of adeno-associated virus (AAV), bovine AAV(b-AAV), canine AAV (CAAV), and avian AAV (AAAV).

Especially preferred are AAVs selected from the group consisting ofAAV-1, AAV-2, AAV-3b, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10,AAV-11, AAV-12 and AAV13, especially AAV-2. AAV1 to AAV12 specifydefined serotypes of adeno-associated virus (AAV).

As described herein in more detail, it is especially preferred that theVP3 cds further comprises at least one mutation. The mutation is incomparison to the respective wildtype parvoviral sequence, preferablyselected from the group consisting of one or more deletion(s), one ormore insertion(s), one or more substitution(s), and a combination ofthese mutations.

It is an embodiment of this invention that the VP3 cds comprises one ormore silent mutation(s). By introducing DNA mutations that do not resultin a change to the AA sequence of the VP3 protein it is possible tooptimize the codon usage of the cds of VP3 e.g. to enhance itsexpression. Due to the degeneracy of the genetic code one AA may bespecified by more then one codon, for example the AA glutamic acid isspecified by GAA and GAG codons. Accordingly, for each AA of thestructural protein VP3 one would select those codons that are translatedwith higher efficiency and mutate the cds respectively. As alreadydiscussed these mutations do not change the AA sequence of the protein,that is why they are called silent, but of course change the nucleotidesequence including the diffusible molecule coded by fragment Z/thenucleic acid of the invention. For this reason it is an especiallypreferred embodiment of this invention that only the part of the VP3 cdsis modified by insertion or optimization of codon usage e.g. to gethigher expression of VP3 in the chosen setup that does not overlap withthe sequence of fragment Z, or in a trans situation as described aboveanywhere within the VP3 cds.

In a preferred embodiment, the one or more mutation(s) of the VP3 cdslead(s) to one or more mutations located on the surface of a VP3 VLP.The surface-located regions of the structural protein can be determinedby analyzing the crystal structure, which is known for AAV2 (Xie et al.,2002). If the crystal structure is still not available for the chosenserotype the chosen VP3 sequence can be aligned to the VP3 sequence ofat least one different serotype with an already known crystal structureto identify homologous regions of interest. The alignment can be doneusing a commercially available software like e.g. Multialign (Corpet,1988) and standard parameters described there.

In a further preferred embodiment, the one or more mutation(s) of theVP3 cds lead(s) to one or more mutation(s) located at the N-terminus ofVP3. Preferably, the N-terminus is defined as the N-terminal 10,preferably N-terminal 5, especially N-terminal 2 amino acids of therespective VP3. Especially preferred is an insertion at or correspondingto an insertion directly N- or C-terminal, preferably directlyC-terminal of AA 203 (I-203).

It is preferred according to this invention that the insertion(s) isinserted into one or more positions selected from the group consistingof I-261, I-266, I-381, I-447, I-448, I-453, I-459, I-471, I-534, I-570,I-573, I-584, I-587, I-588, I-591, I-657, I-664, I-713 and I-716,preferably I-261, I-453, I-534, I-570, I-573 and I-587, especiallyI-587.

The used nomenclature I-### refers to the insertion site with ### namingthe AA number relative to the VP1 protein of AAV-2, however meaning thatthe insertion may be located directly N- or C-terminal, preferablydirectly C-terminal of one AA in the sequence of 5 Aas N- or C-terminalof the given AA, preferably 3, more preferably 2, especially 1 AA(s)N-or C-terminal of the given AA. For parvoviruses other than AAV-2 thecorresponding insertion sites can be identified by performing an AAalignment or by comparison of the capsid structures, if available. Suchalignment has been performed for the parvoviruses AAV-1, AAV-2, AAV-3b,AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-10, AAV-11, b-AAV, GPV, B19, MVM,FPV and CPV (FIG. 3 of WO 2008/145400).

The AA position after which the insertion was introduced and which namedthe site is underlined. It is also possible likewise to introduce aninsertion into the five directly adjacent Aas located next to theunderlined AA, because these are likewise located within a loop in theAAV2 capsid.

For example the insertion site I-587 corresponds to an insertion beforeand/or after one of the following Aas indicated by emphasis:

FQSSS TDPAT of AAV1, LQRGN ₅₈₇ RQAAT of AAV2, LQSSN TAPTT of AAV-3b,LQSSS TDPAT of AAV-6, LQAAN TAAQT of AAV-7, LQQQN TAPQI of AAV-8, LQQANTGPIV of AAV10, NQNAT TAPIT of AAV11 and NQSST TAPAT of AAV-5.

Further, the insertion site I-453 corresponds to an insertion directlyN- or C-terminal of the following ten Aas each, preferably directlyC-terminal of the AA indicated by emphasis

QNQSG SAQNK of AAV-1, NTPSG ₄₅₃ TTTQS of AAV-2, GTTSG TTNQS of AAV-3b,QNQSG SAQNK of AAV-6, SNPGG TAGNR of AAV-7, QTTGG TANTQ of AAV-8, QSTGGTQGTQ of AAV-10, SGETL NQGNA of AAV-11 and FVSTN NTGGV of AAV-5.

Relating to the AAV2 sequence insertion sites for AAV and otherparvoviruses encompassed by this invention are listed Table 1.

TABLE 1 Insertion sites for parvoviruses insertion corresponding siteAA/sequence of AAV2 references I-261 S₂₆₁ YKQIS₂₆₁ SQSGA (Girod et al.,1999) I-266 A₂₆₆ SQSGA₂₆₆ SNDNH (Wu et al., 2000) I-381 N₃₈₁ YLTLN₃₈₁NGSQA (Girod et al., 1999) I-447 R₄₄₇ YYLSR₄₄₇ TNTPS (Girod et al.,1999, Wu et al., 2000) I-448 T₄₄₈ YLSRT₄₄₈ NTPSG (Grifman et al., 2001)I-453 G₄₅₃ NTPSG₄₅₃ TTTQS WO 2008/145400 I-459 R₄₅₉ TTQSR₄₅₉ LQFSQ (Shiet al., 2001, Arnold et al., 2006) I-471 R₄₇₁ ASDIR₄₇₁ DQSRN (Asokan andSamulski, 2006, Moskalenko et al., 2000) I-534 F₅₃₄ EEKFF₅₃₄ PQSGV(Girod et al., 1999) I-570 P₅₇₀ RTTNP₅₇₀ VATEQ I-573 T₅₇₃ NPVAT₅₇₃ EQYGS(Girod et al., 1999) I-584 Q₅₈₄ STNLQ₅₈₄ RGNRQ (Shi et al., 2001, Shiand Bartlett, 2003) I-587 N₅₈₇ LQRGN₅₈₇ RQAAT (Girod et al., 1999, Shiet al., 2001, Maheshri et al., 2006, Ried et al., 2002, Grifman et al.,2001, Nicklin et al., 2001, Arnold et al., 2006) I-588 R₅₈₈ QRGNR₅₈₈QAATA (Shi and Bartlett, 2003) I-591 A₅₉₁ NRQAA₅₉₁ TADVN (Wu et al.,2000) I-657 P₆₅₇ VPANP₆₅₇ STTFS I-664 A₆₆₄ TFSAA₆₆₄ KFASF (Wu et al.,2000) I-713 T₇₁₃ NVDFT₇₁₃ VDTNG I-716 T₇₁₆ FTVDT₇₁₆ NGVYS (Maheshri etal., 2006)

I-570 is especially suitable as an insertion site that goes along with adeletion of given Aas of the parvovirus structural protein at the siteof insertion, leading to a complete substitution. In this case the AasRTTNPVATEQ can be substituted by an epi- or mimotope.

Insertions have been successfully made into AAV-serotypes other thanAAV2.

TABLE 2 Insertions into AAV-serotypes other than AAV2 AAV insertionsite/ serotype sequence AA relative to AAV2 references AAV1 FQSSS₅₈₈TDPAT I-587 N₅₈₇ own data AAV1 SSSTD₅₉₀ PATGD I-589 Q₅₈₉ (Arnold et al.,2006, Stachler and Bartlett, 2006) AAV-3 NNLQS₅₈₆-SNTAP I-585 R₅₈₅(Arnold et al., 2006) AAV-4 GGDQS₅₈₄-NSNLP I-585 (Arnold et al., 2006)AAV-5 TNNQS₅₇₅-STTAP I-585 (Arnold et al., 2006)

The most preferred insertion sites are:

-   i) I-587 as various insertions have been made in the AA stretch    around N₅₈₇ (LQRGN₅₈₇ RQAAT) of AAV2. Within this stretch insertions    of various peptides were made C-terminal of Aas Q₅₈₄, N₅₈₇, R₅₈₈ and    A₅₉₁ in AAV2 and C-terminal of Aas of other AAV-serotypes    corresponding to R₅₈₅ and Q₅₈₉ of AAV2.-   ii) I-453 as epitopes have been successfully inserted C-terminal of    G₄₅₃ in AAV2.-   iii) FQSSS₅₈₈ TDPAT or SSSTD₅₉₀ PATGD of AAV1.-   iv) I-261 as according to this invention epitopes have been    successfully inserted C-terminal of S₂₆₁ in AAV2.-   v) I-534 as according to this invention epitopes have been    successfully inserted C-terminal of F₅₃₄ in AAV2.-   vi) I-570 as according to this invention epitopes have been    successfully inserted C-terminal of P₅₇₀ in AAV2.-   vii) I-573 as according to this invention epitopes have been    successfully inserted C-terminal of T₅₇₃ in AAV2.

Corresponding Aas for all insertion sites specified herein forparvoviruses disclosed herein can be retrieved from the alignment inFIG. 3 of WO 2008/145400. For those parvoviruses not listed therein analignment under standard parameters as used there can be performed withthe provided AA sequence of such parvovirus and the corresponding AA canbe retrieved from such alignment.

According to this invention two insertions may be preferred and are madeinto two positions selected from the group consisting of I-261, I-453,I-534, I-570, I-573 and I-587, preferably I-261 in combination withI-587, I-261 in combination with I-453 or I-453 in combination withI-587. With respect to triple insertions, preferred combinations aremade into three positions of VP3, preferably an insertion in position453 in combination with an insertion in position 587 and in combinationwith an additional mutation, more preferably in positions I-453, I-587combined with one of the I-534, I-570 and I-573.

Particularly for vaccination applications AAV particles presenting theselected epitope have to be generated. Therefore, it is preferred thatthe VP3 cds comprises at least one epitope heterologous to the virus.

It is further preferred that the epitope of the VP3 protein is a B-cellepitope. Preferably the B-cell epitope is a part of an antigen.Preferred antigens are serum proteins, proteins that can be found atleast under certain conditions (e.g. in a disease state) in the blood,membrane proteins, especially receptor proteins (e.g. CD20,acetylcholine receptors, IL13R, EGFR), and surface antigens ofinfectious agents, preferably not immuno-dominant epitopes of suchsurface antigens. Especially preferred antigens are IgE, tumor-antigens(e.g. Melan A, high molecular weight melanoma associated antigen (HMWMAA), CA125, IL13R, Her2/NEU, L1 cell adhesion molecule), VEGF, EGFR,CD20, IL1, IL4, IL5, IL6, IL9, IL13, IL17, IL18, IL33, TSLP (thymicstromal lymphopoietin), CETP (cholesterol ester transfer protein),TNF-family members (e.g. TNF-α), or B-amyloid.

In a further embodiment the VP3 comprises at least one B-cell epitopeheterologous to the parvovirus, which is preferably not identical to apathogen, particularly to a B-cell epitope of a pathogen, wherein theB-cell epitope is located on the surface of the virus. In a preferredembodiment the VP3 is capable of inducing an immunoglobulin capable ofbinding to the antigen the B-cell epitope is derived from.

In a preferred embodiment, the B-cell epitope is inserted into I-453and/or I-587, especially into I-453 and/or I-587 of AAV1, AAV2 or AAV4.

It is especially preferred that an identical B-cell epitope is insertedat two or more different insertion sites, if it is key to have a largenumber of identical peptides being optimally presented on the surface ofa capsid, especially in case direct B-cell receptor crosslinking shouldbe required for T-cell independent priming of B-cells and breaking of Bcell tolerance against self-antigens. A higher density of B-cellepitopes increases the likelihood of optimal peptide-specific B-cellreceptor crosslinking which requires a defined distance between B-cellreceptors, and therefore, respective B-cell epitopes being presented ona parvovirus capsid.

Moreover, a larger number of inserted B-cell epitopes decreases theprobability for undesired immune reactions against the parvovirusbackbone due to i) masking of natural parvovirus B-cell epi-/mimotopesand/or ii) slight structural capsid changes rendering these naturalB-cell epi-/mimotopes less immunogenic. Accordingly, parvovirusstructural proteins comprising at least three insertions are especiallypreferred.

Taken together, preferred insertion sites for superficial presentationof epitopes are the positions following the amino acids that correspondto the AAV2 amino acids number I-261, I-266, I-381, I-447, I-448, I-453,I-459, I-471, I-534, I-570, I-573, I-584, I-587, I-588, I-591, I-657,I-664, I-713 and I-716, especially I-261, I-453, I-534, I-570, I-573,I-587, and I-588, most preferably I-453 and I-587.

In a further embodiment the insertions, whether terminal or internal,are combined with the deletion of one or more amino acids, leading to apartial or 1:1 substitution of amino acids by different amino acids,wherein partial substitution means that e.g. 8 amino acids aresubstituted by 6 different amino acids, and a 1:1 substitution meansthat e.g. 8 amino acids are substituted by 8 different amino acids.

In one embodiment of this invention the VP3 is comprised in a fusionprotein, e.g. fused to a second protein or peptide. In an especiallypreferred embodiment B-cell epitopes in particular epitopes larger than20 amino acids are fused to the N-terminus of VP3.

In one specific embodiment the VP3 comprises at least one tag useful forbinding to a ligand. In an especially preferred embodiment said tag isintroduced in the parvovirus mutated structural protein by a furthermutation. Such tags are well known in the art, Table 3.

TABLE 3 Tags and corresponding ligands Tag Ligand AU1 Anti AU1monoclonal antibody HIS Nickel GST Glutathione Protein A IgG Biotin orStrep Streptavidin Calmodulin-binding peptide Calmodulin Fc-Peptide ofIgG Protein A Flag GLAG- or 3xFLAG peptide HA (hem agglutinin) HApeptide

In another embodiment of the present invention the VP3 comprises atleast one further mutation. The mutation may be any suitable mutation,such as any of those defined above.

For example, one or several further mutation(s) of the VP3 might beadequate to e.g. i) introduce additional or even identical B-cellepitopes of the same target antigen, and/or ii) B-cell epitopes of oneor more further target protein(s) (multi-target vaccine), T-cellepitope(s) to further promote the desired T-cell immune response,peptide sequence(s) to target and/or activate antigen-presenting cells,or to obtain capsid mutants with reduced immunogenicity of the coreparticle. The latter might be one possibility to setup an efficientprime/boost regimen.

Besides, a further mutation of the parvovirus mutated structural proteinat a different position can be used to compose more complex mimotopes,to modify certain properties of the virion, e.g. it can be used tomodify its natural antigenicity (e.g. Huttner et al., 2003, and WO01/05990), to modify its chromatographic properties (e.g. WO 01/05991),to insert a second B-cell epitope, to insert a T-helper epitope, or toinsert a CTL epitope. Such further mutation is selected from a pointmutation, an internal or terminal deletion, an insertion and asubstitution. Preferably, the further (second) insertion is internallye.g. by an N- or C-terminal fusion.

Another aspect of the invention is a parvoviral particle obtainable fromany of the methods disclosed above. Based upon the above describedmethods we were able to produce parvoviral particles which essentiallyconsist only of VP3 and do not comprise a heterologous nuclearlocalization signal (NLS). Such particles do not contain Rep protein,particularly Rep40, Rep52, Rep68 and Rep78. The described methods enablethe production of sufficient quantities/yields for the manufacture ofmedicaments in a commercial scale.

Another aspect of the invention relates to a parvoviral particleconsisting essentially of VP3, wherein the VP3 optionally comprises oneor more mutation(s) as compared to the corresponding wildtype VP3, andwherein the VP3 does not contain a heterologous nuclear localizationsignal (NLS), and wherein the particle does not contain Rep protein,particularly functional Rep40, Rep52, Rep68 and Rep78.

Especially preferred is a parvoviral particle wherein the capsidconsists only of VP3.

With respect to the one or more mutations it is referred to themutations as described before.

One further aspect of the invention is an expression cassette Acomprising a VP3 cds as defined before and a heterologous promoterthereto, wherein transcription of VP3 is driven by the heterologouspromoter, and wherein the expression cassette is capable of expressingessentially only VP3. Especially preferred are expression cassettes thatexpress only VP3. The construct is used to express mutated VP3 accordingto this invention. The sequence of VP3 can be mutated as described. Asunderstood by the skilled person the expression cassette furthercomprises a poly(A) sequence.

Another aspect of the invention is an expression cassette B comprising afragment Z as defined before and a promoter heterologous thereto,wherein transcription of fragment Z is driven by the heterologouspromoter. The construct is used to express fragment Z according to thisinvention. The sequence of fragment Z can be mutated as described.Optionally, this expression cassette further comprises a poly(A)sequence.

A further aspect of the invention is an expression cassette C comprising(i) a VP3 cds as defined before and fragment Z as defined before, and(ii) a promoter heterologous thereto, wherein the expression of VP3 andfragment Z is driven by this one heterologous promoter.

In a further aspect of the invention the expression cassette comprisinga heterologous promoter, a VP3 cds as described above and the nucleicacid of the invention are combined, wherein the expression of VP3 and ofthe polypeptide encoded by the nucleic acid (AAP or AAP variant) isdriven by this one heterologous promoter. Optionally, this expressioncassette further comprises a poly(A) sequence.

It is one further aspect that at least one expression cassette A/VP3expression cassette and at least one expression cassette B/expressioncassette comprising the nucleic acid of the invention are combined in akit. By combining expression of parvoviral VP3 from the VP3 cds andexpression of fragment Z/the nucleic acid of the invention it ispossible to generate particles consisting essentially only of VP3according to this invention.

Another aspect relates to a kit comprising at least one VP3 expressioncassette A and at least one nucleic acid of the invention or a kitcomprising at least one expression cassette C for the combined andsimultaneous expression of VP3 and fragment Z in the cell and generationof VP3 VLPs. Such kits preferably additionally contain a manual.

Still another aspect of the present invention relates to a medicamentcomprising the parvovirus particle according to the invention.Medicaments according to the present invention have numerous advantagesover the prior art. The immune system of a mammal is specialized togenerate strong antibody responses against viral capsid proteins due tothe co-evolution of mammals and their immune system on one hand andviruses on the other hand. Strong antibody responses means titers of1,000 to >100,000 measured in a standard ELISA. Virus-like particles arehighly immunogenic due to resemblance of a virus, the repetitive andhighly structural pattern of antigens, and efficient uptake of suchparticles by antigen-presenting cells. The size of the virion, thedensity and symmetric order of B-cell epitopes and the optimal distanceof about 50 to 100 Å between any two B-cell epitopes plays a major roleregarding very strong T-cell independent B-cell responses mediated bydirect cross-linking of the respective B-cell receptor breaking evenB-cell tolerance against self-antigens or tolerogens (Szomolanyi-Tsudaand Welsh, 1998, Szomolanyi-Tsuda et al., 1998, Szomolanyi-Tsuda et al.,2000, Szomolanyi-Tsuda et al., 2001, Zinkernagel, 2002, Bachmann et al.,1993).

Taken together, such medicaments are capable of inducing a polyclonalimmune response against certain B-cell epitopes that leads to an activeimmune response resulting in high and long lasting antibody titers. Themultimeric structure of the virion contains a large number of denselypacked identical epitopes directly cross-linking the respective receptoron B-cells and, thereby, inducing a T-cell independent B-cell response.The particulate structure of the medicament further supports the immuneresponse via efficient uptake by antigen-presenting cells which activateT-cells finally triggering IgG class switch and hypermutation ofactivated B-cells, leading to the persistent release of high-affinityIgG antibodies and differentiation of B-cells into memory cells.

Using the methods of the current invention such medicaments can easilybe produced.

The medicament of the present invention may further additionallycomprise one or more excipients. The excipient is a pharmaceuticallyacceptable carrier and/or excipient.

Excipients are conventional and may include buffers, stabilizers,diluents, preservatives, and solubilizers. Remington's PharmaceuticalSciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 15^(th)Edition (1975), describes compositions and formulations suitable forpharmaceutical delivery of the (parvo)viral particles herein disclosed.In general, the nature of the carrier or excipients will depend on theparticular mode of administration being employed. For instance,parenteral formulations usually comprise injectable fluids that includepharmaceutically and physiologically acceptable fluids such as water,physiological saline, balanced salt solutions, aqueous dextrose,glycerol or the like as a vehicle. For solid compositions (e. g. powder,pill, tablet, or capsule forms), conventional non-toxic solid carrierscan include, for example, pharmaceutical grades of mannitol, lactose,starch, or magnesium stearate. In addition to biologically neutralcarriers, pharmaceutical compositions to be administered can containminor amounts of non-toxic auxiliary substances, such as wetting oremulsifying agents, preservatives, and pH buffering agents and the like,for example sodium acetate or sorbitan monolaurate.

In a further embodiment the medicament is a vaccine. In general, avaccine is a preparation consisting of antigens of a disease-causingagent which, when introduced into the body, stimulates the production ofspecific antibodies or altered cells. This produces generally an immuneresponse as an active principle. Particularly, the parvovirus particlesassembled of VP3 comprise at least one B-cell epitope heterologous tothe parvovirus, preferably for preventing or treating an autoimmunedisease (e.g. diabetes type 1), a tumor disease (examples are: melanoma:e.g. HMW MAA, glioblastome multiforme: e.g. CA125, anti-IL13R, coloncancer: e.g. CA125 or anti-EGF AND/OR, breast cancer: e.g. Her2/NEU,ovarian cancer: e.g. L1 adhesion molecule, B-cell lymphoma: e.g. CD20),an allergic disease (asthma, allergies such as allergic rhinitis,examples for targets are IgE, IL4, IL5, IL9, IL13, IL18, IL33, TSLP), ametabolic disease (e.g. high cholesterol, intervention into thecholesterol metabolism (target example: CETP), obesity, hypertension(target example angiotensin II), an inflammatory disease (e g.rheumatoid arthritis, Crohn's disease, target examples' IL6, IL17 andTNF-α), a neurological disease (e.g. Alzheimer's disease; targetexample: 3-Amyloid) or to be used in ophthalmology (e.g. AMD, targetexample VEGF).

Also encompassed by the present inventions are methods for vaccinationand/or for treating or preventing the diseases specified herein byadministering to a patient an effective amount of parvovirus particlesof the invention and/or expression constructs coding for the VP3 VLP ofthe invention.

In a preferred embodiment the vaccine further comprises one or moreadjuvants, particularly as an immunostimulatory substance. The adjuvantcan be selected based on the method of administration and may includemineral oil-based adjuvants such as Freund's complete and incompleteadjuvant, Montanide incomplete Seppic adjuvant such as ISA, oil in wateremulsion adjuvants such as the Ribi adjuvant system, syntax adjuvantformulation containing muramyl dipeptide, or aluminum salt adjuvants.

One embodiment of this invention is a medicament for the prevention ortreatment of an autoimmune disease, an infectious disease, a tumordisease, an allergic disease, a metabolic disease, a (chronic)inflammatory disease, a neurological disease, addiction or to be used inophthalmology. Preferred autoimmune diseases and/or a chronicinflammatory diseases are rheumatoid arthritis, psoriasis and Crohn'sdisease. A preferred tumor disease is a disease eligible for treatmentwith a monoclonal antibody, e.g. trastuzumab. Preferred allergicdiseases are asthma and allergies, e.g. allergic rhinitis. Examples forpreferred allergens are birch pollen, house dust mite and grass pollen.A preferred neurological disease is Alzheimer's disease. A preferredmetabolic disease is atherosclerosis. A preferred ophthalmologicaldisease is age-related macular degeneration. In a further preferredembodiment the parvovirus particle or the medicament is used in a methodof breaking B-cell tolerance, meaning inducing antibodies against aself-antigen.

In a further special embodiment the disease treated by the medicament isnot an infectious disease.

Moreover, the parvovirus mutated structural protein of the medicament isnot used as a vector in gene therapy.

In another embodiment the parvoviral particle of the invention is usedfor gene therapy.

According to this invention an embodiment is the use of a parvovirusparticle as defined above, preferably the medicament as defined above,comprising at least one B-cell epitope heterologous to the parvovirusfor the manufacture of a vaccine, preferably for preventing or treatingan autoimmune disease and/or a chronic inflammatory disease, preferablyrheumatoid arthritis and/or Crohn's disease, a tumor disease, anallergic disease, asthma, Alzheimer's disease, atherosclerosis, ametabolic disease, an inflammatory disease, a neurological disease or tobe used in ophthalmology.

In this document, the content of all cited documents is included byreference.

The following examples and figures are intended to explain the inventionin detail without restricting it.

FIGURES

FIG. 1: Schematic organization of the AAV capsid gene.

The coding DNA for the cap gene is shown in the first line, the Capproteins VP1, VP2 and VP3 in the following ones. Nucleotide numberscorrespond to the genome sequence of AAV-2 given by Ruffing et al.(1994) accessible from NCBI (number of entree: NC_001401). Numbering ofamino acid (AA) sequences according to VP1 of AAV2 (Girod et al. 1999).EcoNI and BsiWI restriction sites are marked. Not to scale.

FIGS. 2A-2D: Nucleotide sequences of fragment Z of different AAVs.

The nucleotide sequences of fragment Z of the parvoviruses AAV1(NC_002077), AAV2 (AF043303), AAV3b (AF028705), AAV4 (U89790), AAV5(NC_006152), AAV6 (AF028704), AAV7 (AF513851), AAV8 (AF513852), AAV10(AY631965), AAV11 (AY631966), and b-AAV (NC_005889) are given (numbersof nucleotide entrees according to NCBI are given in brackets). +1indicates the position of the first nucleotide coding for the ATG startcodon of VP3. The 44 nucleotides upstream and 242 nucleotides downstreamof the +1 position are shown. The ATG start codon of VP3 is underlined.

FIG. 3: Schematic representation of the different expression constructssuitable for assembly of VP3 particles.

Six possible expression constructs differing in the set-up of thefragment Z sequence and VP3 cds are shown by different boxes asindicated. In the cis situation they are expressed under the same onepromoter whereas in trans two separate promoters drive their expression,as indicated by the circle. +1 indicates the position of the firstnucleotide coding for the ATG start codon of VP3. The DNA of fragment Zcomprising at least 44 nucleotides upstream and more than 242nucleotides downstream of the +1 position are boxed (compare FIGS.2A-2D). +1602 marks the number of the last nucleotide of the TAA stopcodon at the 3′ end of the VP3 cds (as outlined in FIG. 1). An arbitrarynumber of nucleotides can separate the VP3 cds and fragment Z and ismarked by //. Not to scale.

FIG. 4: Schematic organization of the rep and cap genes, as well asposition of different restriction sites used for cloning of expressionconstructs.

Schematic representation of the rep and cap genes in the parvovirusgenome. The position of the restriction sites R1 to R5 used for cloningof the different expression constructs, as well as the positions of thetranslation start codons of the three capsid proteins are marked. Not toscale

FIGS. 5A-5D: Comparison of capsid assembly using different VP proteinexpression constructs.

FIGS. 5A and 5B) Schematic representation of the cap gene expressionconstructs used for analysis of VP protein expression and to studycapsid assembly. Plasmids pCMV-VP3/1882 to pCMV-VP3/2809 are derivedfrom plasmid pVP3. Numbers indicate nucleotide positions in the AAV2genome according to Ruffing et al., 1994 (supra). Arrows representtranslation start sites of the VP proteins, mutated translation startsites are labeled with a cross. The ability of the proteins expressedfrom these expression constructs to assemble capsids is given in theright column (corresponding to the quantification in FIG. 5D, ++corresponds to peak titer of capsids, − means that no capsids could bedetected, + means that capsid assembly is detectable. FIG. 5C) Westernblot analysis of expressed VP proteins was performed using antibody B1which detects all three capsid proteins or antibody A69 which detectsonly VP1 and VP2. In each lane a different expression construct isseparated, name according to FIGS. 5A and 5B. The position of the threecapsid proteins is marked. FIG. 5D) Capsid formation was quantified byan ELISA based on monoclonal antibody A20. Means+/−standard deviationsof at least three independent experiments are shown; asterisk indicatesconstructs for which no capsids could be detected.

FIGS. 6A-6D: Complementation of VP3 capsid assembly by VP2N-gfp.

FIG. 6A) Schematic representation of the fusion construct, pVP2N-gfp, aswell as of its transcripts VP2N-gfp, VP3N-gfp and GFP as indicated. FIG.6B) Western blot detection of VP3 (B1 antibody), VP3N-gfp fusion protein(anti-gfp antibody) and VP2N-gfp (A69 antibody) expression in HeLa cellsafter co-transfection of pVP3/2809 (1) and decreasing amounts ofpVP2N-gfp (1, 1/5, 1/50, 1/500, del meaning 0) as indicated. FIG. 6C)Detection of capsid formation by indirect immunofluorescence usingantibody A20 in HeLa cells co-transfected with pVP3/2809 and pVP2N-gfpin different ratios as marked and shown in FIG. 6B. D) Quantification ofcapsid formation in HeLa cells co-transfected with pVP3/2809 andpVP2N-gfp in different ratios using the A20 based capsid ELISA. Again,the different plasmid ratios are marked and correspond to those shown inFIGS. 6B and 6C. For each experiment the mean concentration ofcapsids+/−standard deviations of at least two independent experimentsare shown; asterisk indicates samples for which no capsids could bedetected.

FIG. 7: Substoichiometric incorporation of truncated VP2 within VP3particles in the cis situation.

Western blot analysis of purified wt AAV and capsids derived frompVP3/2696 or pVP3/2809 trans-complemented with pVP2N-gfp. Detection ofVP1 and VP2 occurred with antibody A69. Different amounts of capsids asindicated were loaded to the gel for a qualitative estimation of theratio of different signals (VP2tru=truncated VP2).

FIGS. 8A-8F: Characterization of helper plasmid pVP2Ncm-gfp withalternative codon usage.

FIGS. 8A-8C) Alignment of wt (VP2N, SEQ ID NO: 145) and codon modifiedVP2N (VP2Ncm, SEQ ID NO: 146) DNA sequences of the respective constructspVP2N-gfp (details in FIG. 6A) and pVP2Ncm-gfp.

FIG. 8D) Western blot of 293-T cell extracts after transfection of theindicated plasmids with monoclonal antibody A69. FIG. 8E) Fluorescenceimages of HeLa cells transfected with pVP2N-gfp: The upper and lowerleft panels represent total GFP fluorescence. The upper and lower rightpanels show indirect immunofluorescence of the VP2 part within VP2N-gfpvisualized by the A69 antibody and the respective secondary Cy3-labeledgoat anti-mouse antibody. FIG. 8F) Quantification of capsid formation in293-T cells co-transfected with pCMV-VP3/2809 and the indicated plasmidsusing the A20 based capsid ELISA. Means+/−standard deviations of atleast three independent experiments are shown; asterisk indicates samplefor which no capsids could be detected.

FIGS. 9A-9C: Stop codon mutagenesis within the trans-complementationconstruct

FIG. 9A) Schematic representation of pVP2N-gfp constructs withtranslation stop codons in the VP2N reading frame at four differentpositions. Numbers of the substituted nucleotides refer to thenucleotide positions of the AAV2 genome. In pVP2N/stopA the cag-codonstarting at nucleotide 2770 and coding for glutamine has been mutatedinto tag, in pVP2N/stopB the gga-codon starting at nucleotide 2797 andcoding for glycine has been mutated into tga, in pVP2N/stopC theagt-codon starting at nucleotide 2821 and coding for serine has beenmutated into tga, and in pVP2N/stopD the gga-codon starting atnucleotide 2878 and coding for glycine has been mutated into tga. FIG.9B) Western blot of 293-T cell extracts after co-transfection ofpCMV-VP3/2809 and the indicated plasmids with monoclonal antibodies B1and A69. FIG. 9C) Quantification of capsid formation in 293-T cellsco-transfected with pCMV-VP3/2809 and the indicated plasmids using theA20 based capsid ELISA. Means+/−standard deviations of at least threeindependent experiments are shown; asterisk indicates sample for whichno capsids could be detected.

FIG. 10: Cellular localization of capsid proteins and capsids obtainedby expression of different cap gene mutants.

Localization of capsid proteins expressed from different constructs inHeLa cells was visualized by double immunofluorescence using apolyclonal rabbit antiserum detecting total capsid proteins (VPs) andmonoclonal antibody A20 detecting assembled capsids. The transfectedplasmids are indicated at the left margin.

Immunofluorescence staining of transfected HeLa cells with the A20antibody showed that the VP protein of mutant pCMV-VP3/2696RKR168-170AAAwas as efficient in capsid assembly as wt AAV. For the constructpCMV-VP3/2696RKR168-170AAA the postulated NLS was mutated by convertingthe RKR peptide (AA 168-170).

FIGS. 11A-11C: Capsid assembly of VP3 modified by a NLS or an N terminalextension of human serum albumin.

FIG. 11A) Schematic representation of NLS-VP3 and HSA-VP3 used foranalysis of capsid assembly. FIG. 11B) Immuno dot blot analysis offractions obtained from COS-1 cell extracts separated on sucrosegradients. The cells were harvested 48 h post transfection of theplasmids indicated in the left margin. Note that reaction with the A20antibody was performed under non-denaturing conditions to detectassembled capsids, whereas reaction with B1 antibody was performed afterdenaturation of the capsids to detect single capsid proteins. Thesedimentation constant of the viral capsid is indicated (60 S). FIG.11C) Indirect double immunofluorescence of HeLa cells transfected withplasmids indicated above the images using a polyclonal VP antiserum(VPs) to localize total expressed capsid proteins (upper row) andantibody A20 to detect assembled capsids (lower row). VP2N-egfp is asynonym for pVP2N-gfp.

FIGS. 12A-12C: VP3 particle production in insect cells

FIG. 12A) Schematic representation of constructs used for AAV productionin insect cells. FIG. 12B) Western blot analysis of expressed VPproteins was performed using antibody SA7885 (1:10000 dilution) apolyclonal rabbit serum that detects all three capsid proteins andsubsequent the secondary antibody anti rabbit IgG-HRP 1:2500 (Dianova,Hamburg, Germany).

FIG. 12C) Capsid formation was quantified by an ELISA based onmonoclonal antibody A20. Means+/−standard deviations of 2 (VP2construct) or 4 (VP3 and VP1_Mod4) independent experiments are shown.

FIG. 13: Western Blot analyses of different AAV1 constructs

Western blot analysis of expressed VP proteins in crude lysates of 293cells transfected with different AAV1 constructs: pCI_VP2/2539_AAV1,pCI_VP3/2539_AAV1 mutACG, pCI_VP3/2634_AAV1 mutACG and pUCAV1. Detectionof VP proteins was performed using the B1 antibody (dilution: 1:250)(Progen Heidelberg, Germany) and subsequent the secondary antibody antimouse IgG-HRP 1:2500 (Dianova, Hamburg, Germany).

2E10 particles per construct were loaded according to AAV1 titration byan AAV1 capsid ELISA (Progen Heidelberg, Germany).

The Western Blot shows that construct pUCAV1 expresses the three capsidproteins VP1, VP2 and VP3 (lane 5) whereas pCI_VP2/2539_AAV1 leads toexpression of VP2 and VP3 (lane 2) and within lysates of cellstransfected with pCI_VP3/2539_AAV1 mutACG and pCI_VP3/2634_AAV1 mutACGonly VP3 could be detected (lane 3 and 4).

FIG. 14: Trans-complementation of an AAV1 VP2 construct with pVP2N-gfpof AAV2

Western blot analysis of cell extracts transfected with VP3 expressionconstruct of AAV2 pCMV-VP3/2809 or of AAV1 pCMV-AAV1VP3/2828 (indicatedin the figure as AAV2 or AAV1, respectively) with or withoutcotransfection of pVP2N-gfp. AAV1 and AAV2 VP3 was detected by theantibody B1 (Progen, Heidelberg, Germany) which recognizes an epitopecompletely conserved between AAV1 and AAV2. The VP2N-gfp protein wasdetected by antibody A69 (Progen, Heidelberg, Germany).

FIGS. 15A-15C: Comparison of particle production efficiency usingdifferent pCMV-VP expression vectors

FIG. 15A) Schematic representation of constructs. pCI-VP, pCI-VP2 andpCI-VP3 were cloned by PCR amplification of the respective VP codingregions using primer with XhoI (5′-) and NotI (3′-) overhangs andsubcloning of the XhoI-/NotI-digested PCR products into theXhoI-/NotI-digested vector pCI (PROMEGA). In case of pCI-VP2, the startcodon for VP2 was changed from ACG to ATG at the same time.

For cloning of the constructs pCI-VP2mutACG, pCMV-NLS-VP3, andpCMV-VP3/2696 please refer to elsewhere.

FIG. 15B) For transfection 5.0E+05 293-T cells were seeded into eachwell of a 6-well cell culture plate in a total volume of 3 ml medium(DMEM containing 10% FCS and ABAM). Cells were cultivated at 37° C. and5% CO₂ in a humidified atmosphere for 24 h. Subsequently cells weretransfected using the calcium phosphate transfection protocol asdisclosed in US 2004/0053410. Briefly, for transfection of one well with293-T cells 6 μg of the indicated plasmids (pCI-VP, pCI-VP2, pCI-VP3,pCI-VP2 and pCI-VP3 in a 1:10 molar ratio, pCMV-NLS-VP3, pCI-VP2mutACG,and pCMV-VP3/2696, respectively) were mixed in 150 μl 270 mM CaCl₂. 150μl 2×BBS (50 mM BES (pH 6.95), 280 mM NaCl and 1.5 mM Na₂HPO₄) was addedto the mixture and the resulting solution was carefully mixed bypipetting. The solution was incubated for 20 min at room temperature andthen added drop-wise to the cells. Cells were incubated at 35° C., 3%CO₂ in a humidified atmosphere for 18 h. After 18 h at 35° C. and 3% CO₂cells were cultivated for an additional 3d at 37° C., 5% CO₂ in ahumidified atmosphere.

Subsequently, 293-T cells were lysed in the medium by three rounds offreeze (−80° C.) and thaw (37° C.) cycles. The lysate (3 ml totalvolume) was cleared by centrifugation and the VLP capsid titer wasdetermined using a commercially available ELISA (AAV Titration ELISA,Progen). Average values of 4 to 6 independent transfections perconstruct are indicated with respective error bars.

Notably, particle production efficacy with construct pCMC-NLS-VP3 wasbelow the detection limit (about 1E+09/ml) and, therefore, at least 3-4logs lower compared to the best VP3 particle production vectorsdescribed in this invention (pCI-VP2mutACG and pCMV-VP3/2696).

FIG. 15C) For transfection 5.0E+05 293-T cells were seeded into eachwell of a 6-well cell culture plate in a total volume of 3 ml medium(DMEM containing 10% FCS and ABAM). Cells were cultivated at 37° C. and5% CO₂ in a humidified atmosphere for 24 h. Subsequently cells weretransfected using the calcium phosphate transfection protocol asdisclosed in US 2004/0053410. Briefly, for transfection of one well with293-T cells 6 μg of the indicated plasmids (pCI-VP, pCI-VP2, pCI-VP3,pCI-VP2 and pCI-VP3 in a 1:10 molar ratio, pCMV-NLS-VP3, pCI-VP2mutACG,and pCMV-VP3/2696, respectively) were mixed in 150 μl 270 mM CaCl₂. 150μl 2×BBS (50 mM BES (pH 6.95), 280 mM NaCl and 1.5 mM Na₂HPO₄) was addedto the mixture and the resulting solution was carefully mixed bypipetting. The solution was incubated for 20 min at room temperature andthen added drop-wise to the cells. Cells were incubated at 35° C., 3%CO₂ in a humidified atmosphere for 18 h. After 18 h at 35° C. and 3% CO₂cells were cultivated for an additional 3d at 37° C., 5% CO₂ in ahumidified atmosphere.

Subsequently, supernatant of 293-T cells was removed, cells were rinsedwith PBS and finally lysed in 300 μl RIPA buffer (25 mM Tris.Cl pH 7.4,150 mM NaCl, 1% IGEPAL, 1% Na.DOC, 0.1% SDS). 100 μl 3×Geba samplebuffer (Gene Bio-Application Ltd) and 25 mM DTT were added, and sampleswere heated at 95° C. for 10 min. Samples were centrifuged and 30 μlcleared supernatant were subjected to SDS page (10% GeBa gels, GeneBio-Application Ltd). Proteins were transferred to a nitrocellulosemembrane (1 h, 230 mA) which was blocked for 1 h at RT subsequently. VPproteins were detected with the antibody B1 (Progen) by overnightincubation at 4° C. in blocking buffer (1:500 dilution), subsequentwashing and incubation with secondary antibody (anti-mouse IgG-HRP;1:2500 in blocking buffer). Finally, the membrane was rinsed again andincubated with super signal pico west substrate (Pierce) for 5 min atRT. AAV capsid proteins are expressed as expected from the different VPexpression vectors.

FIG. 16: Schematic organization of the AAV capsid gene.

The position of ORF2 and the encoded protein AAP is shown in relation tothe position of translation start codons of the Cap proteins VP1, VP2and VP3, as well as the EcoNI and BsiWI restriction sites (as given anddescribed in more detail in FIG. 1). The arrows mark the translationstart site and indicate that VP1, VP2 and VP3 are translated from thesame one reading frame (named first ORF, ORF1, herein) of the cap gene,whereas AAP is translated from a different reading frame (ORF2). ForVP1, VP2 and VP3 the well-defined numbers of the translation startpoints are given.

FIG. 17: Nucleotide sequence of ORF2 and protein sequence of AAP ofAAV2.

The nucleotide sequence of ORF2 of AAV2 (NCBI entrée number NC_001401)from position to 3343 (including the tga stop codon), as well as therespective protein sequence of AAP obtained upon translation of ORF2starting with the first nucleotide of ORF2 is given. marks thenucleotide position of the ATG start codon of VP3 which is underlinedand given in bold. The predicted AAP translation initiation codon CTGcoding for L (leucine) also is underlined and marked in bold.

FIGS. 18A and 18B: Sequence of ORF1cm and ORF2cm.

FIG. 18A) DNA sequence of the codon modified EcoNI-BsiWI restrictionfragment ORF1 cm. FIG. 18B) DNA sequence of the codon modifiedEcoNI-BsiWI restriction fragment ORF2 cm. Translation start codons ofVP2 and VP3 are underlined. Start of ORF2 is marked (1) and position ofthe predicted non-canonical AAP translation initiation codon CTG intactin ORF2 cm is highlighted by a frame. And/orote that the translationstart codon of AAP is mutated into CCG in ORF1cm.

FIGS. 19A-19C: Trans-complementation of VP3 expressing plasmid withpVP2N-gfp.

FIG. 19A) Schematic representation of construct pVP2N-gfp, containingthe EcoNI-BsiWI fragment derived of the AAV2 genome and a gfp-cassette,

FIG. 19B). pVP2N-gfp was co-transfected with pCMV-VP3/2809 in decreasingamounts into 293-T cells, starting with equimolar ratios, in order tocomplement VP3 expression of plasmid pCMV-VP3/2809. For comparison emptyvector pBS (commercially available Bluescript vector) or plasmidpCMV-VP3/2696 were transfected. Samples were analyzed by Western blotusing monoclonal antibodies B1 for detection of VP3 and A69 fordetection of VP2N-gfp and VP2tru (truncated VP2).

FIG. 19C) Capsid formation was quantified by an ELISA based onmonoclonal antibody A20. Means+/−standard deviations of at least threeindependent experiments are shown; asterisks indicate samples for whichno capsids could be detected.

FIGS. 20A-20C: Trans-complementation of VP3 expressing plasmid withpVP2N/ORF1cm and pVP2N/ORF2 cm.

Same experimental setup as described in FIGS. 19A-19C with thedifference that the constructs pVP2N/ORF1cm and pVP2N/ORF2 cm have beenused for trans-complementation. Codon modified DNA sequences (detailedsequences are given in FIG. 18) are represented as shaded boxes in A).

FIGS. 21A-21C: Trans-complementation of VP3 expressing plasmid withpORF2/CTG-AU1, pORF2/ATG-AU1 and pORF2/TTG-AU1.

Same experimental setup as described in FIGS. 19A-19C with thedifference that the constructs pORF2/CTG-AU1, pORF2/ATG-AU1 andpORF2/TTG-A have been used for trans-complementation. They comprise theentire ORF2 of the cap gene (as given in FIG. 17) fused to sequencescoding for an AU1-tag. The predicted AAP translation initiation codon(CTG) was additionally mutated to ATG and TTG

Monoclonal antibody anti-AU1 for detection of AAP-AU1 or polyclonalanti-AAP serum for detection of AAP-AU1 or C-terminally truncated AAP(AAPtru).

FIGS. 22A-22C: Trans-complementation of VP3 expressing plasmid withpVP2N/ORF2stopA, pVP2N/ORF2stopB, and pVP2N/ORF2stopC.

Derivates of pVP2N-gfp harbouring stop codons in ORF2 of the cap genefragment were co-transfected with VP3 expression plasmid pCMV-VP3/2809into 293-T cells.

FIG. 22A) Schematic representation of the constructs pVP2N/ORF2stopA,pVP2N/ORF2stopB, and pVP2N/ORF2stopC, respectively, containing stopcodons in ORF2 of the cap gene fragment at the indicated positions. InpVP2N/ORF2stopA the tgg-codon starting at nucleotide has been mutatedinto tag, in pVP2N/ORF2stopB the caa-codon starting at nucleotide hasbeen mutated into taa, and in pVP2N/ORF2stopC the gaa-codon starting atnucleotide 2879 has been mutated into tga. All mutations do not disruptORF1.

FIG. 22B) Samples were analyzed by Western blot using monoclonalantibodies B1 for detection of VP3 and A69 for detection of VP2N-gfp.

FIG. 22C) Capsid formation was quantified by an ELISA based onmonoclonal antibody A20. Means+/−standard deviations of at least threeindependent experiments are shown; asterisks indicate samples for whichno capsids could be detected.

FIGS. 23A-23D: Trans-complementation of full length AAV2 genomedeficient in AAP expression with different constructs.

FIG. 23A) Schematic representation of plasmid pTAV2.0, harbouring thewildtype AAV2 genome and of plasmid pTAV/ORF1cm, containing the ORF1codon modified EcoNI/BsiWI fragment of the cap gene (shaded box).

FIG. 23B) Plasmids were co-transfected with the indicated constructsinto 293-T cells. Western blot analysis of VP protein expression wasperformed using monoclonal antibody B1. AAP and truncated AAP (AAPtru)were detected with polyclonal anti-AAP serum.

FIGS. 23C and 23D) Capsid formation upon co-transfection of plasmids asindicated in 293-T cells was quantified by an ELISA based on monoclonalantibody A20. Means+/−standard deviations of at least three independentexperiments are shown; asterisks indicate samples for which no capsidscould be detected.

FIGS. 24A-24C: Trans-complementation of full length AAV2 genomecontaining a stop codon in ORF2 of the cap gene by wt genome.

FIG. 24A) Schematic representation of plasmid pTAV2.0, harbouring the wtAAV2 genome and of plasmid pTAV/ORF2stopB, containing a stop codon inORF2 of the cap gene (equivalent position as in plasmid pVP2N/ORF2stopB,FIGS. 22A-22C).

FIG. 24B) Plasmids were co-transfected with empty vector pBS or withpVP2N-gfp (as indicated) into 293-T cells. Western blot analysis of VPprotein expression was performed using monoclonal antibody B1. AAP andAAPtru were detected with polyclonal anti-AAP serum.

FIG. 24C) Capsid formation upon co-transfection of plasmids as indicatedin 293-T cells was quantified by an ELISA based on monoclonal antibodyA20. Means+/−standard deviations of three independent experiments areshown; asterisk indicates sample for which no capsids could be detected.

FIGS. 25A-25C: Immunofluorescence images for intracellular localizationof VP3 and NoLS-VP3, as well as assembled capsids.

FIG. 25A) Schematic representation of the construct used for expressionof VP3 fused to the nucleolar localization signal of HIV Rev (NoLS-VP3)in comparison to the construct expressing NLS-VP3 due to fusion of VP3to the nuclear localization signal of the SV40 large T-antigen (as usedin FIGS. 11A-11C).

FIG. 25B) Indirect double immunofluorescence of HeLa cells transfectedwith plasmids indicated at the left using a polyclonal VP antiserum(VPs) to localize total expressed capsid proteins (left images) andantibody A20 to detect assembled capsids (right images).

FIG. 25C) Indirect double immunofluorescence of HeLa cells transfectedwith plasmids indicated at the left using a monoclonal antibody againstthe AU1-tag (anti-AU1) to localize expressed AAP (left image) andpolyclonal Fibrillarin antibody (anti-Fibrillarin) as a marker fornucleoli localization (middle image). On the right the phase contrastimage of the same sector is shown.

FIGS. 26A-26B: Expression and capsid assembly activity of VP3, NLS-VP3and NoLS-VP3.

FIG. 26A) Western blot analysis of extracts of 293-T cells expressingVP3 or VP3 fusion proteins as indicated was performed using monoclonalantibody B1.

FIG. 26B) Capsid formation in 293-T cells was quantified by an ELISAbased on monoclonal antibody A20. Means+/−standard deviations of atleast three independent experiments are shown; asterisks indicatesamples for which no capsids could be detected.

FIGS. 27A and 27B: Comparison of parvovirus AAP sequences.

Alignment of predicted AAP protein sequences derived from ORF2 of thecap gene of different parvoviruses. Conserved amino acids that are 100%identical in at least 60% of aligned sequences are represented as linesin the lower row. Position of the predicted AAV2 AAP translation startis highlighted by a frame. Non-translated sequences upstream of thepotential translation initiation codons are included as well. NCBIentrée numbers of the corresponding DNA sequences are listed in table 8.

FIG. 28: EM analysis of AAV2 empty particle preparations

Virus-like particles assembled of VP1, VP2 and VP3 (VP1,2,3 VLP) orassembled only of VP3 (VP3 VLP) as indicated.

FIG. 29: Capsid assembly upon trans-complementation

Capsid formation in 293-T cells from constructs pCMV_VP3/2809 of AAV2(AAV2-VP3), pCMV_AAV1VP3/2829 from AAV1 (AAV1-VP3) and a correspondingAAV5 VP3 construct (AAV5-VP3) co-transfected with pVP2N-gfp from AAV2,AAV1 and AAV5 as indicated was quantified by an ELISA based onmonoclonal antibody A20. Bluescript vector (pBS) was used as negativecontrol. Asterisks indicate samples for which no capsids could bedetected.

AMINO ACID SEQUENCES

SEQ ID NO: 1 ILVRLETQTQ YLTPSLSDSH QQPPLVWELI RWLQAVAHQW QTITRAPTEWVIPREIGIAI PHGWATESSP PAPEPGPCPP TTTTSTNKFP ANQEPRTTIT TLATAPLGGILTSTDSTATF HHVTGKDSST TTGDSDPRDS TSSSLTFKSK RSRRMTVRRR LPITLPARFRCLLTRSTSSR TSSARRIKDA SRRSQQTSSW CHSMDTSP SEQ ID NO: 2 SSRHKSQTPPRASARQASSP LKRDSILVRL ATQSQSPIHN LSENLQQPPL LWDLLQWLQA VAHQWQTITKAPTEWVMPQE IGIAIPHGWA TESSPPAPAP GPCPPTITTS TSKSPVLQRG PATTTTTSATAPPGGILIST DSTATFHHVT GSDSSTTIGD SGPRDSTSNS STSKSRRSRR MMASQPSLITLPARFKSSRT RSTSFRTSSA LRTRAASLRS RRTCS SEQ ID NO: 3 ISVRLATQSQSQTLNLSENH QQPPQVWDLI QWLQAVAHQW QTITRVPMEW VIPQEIGIAI PNGWATESSPPAPEPGPCPL TTTISTSKSP ANQELQTTTT TLATAPLGGI LTLTDSTATS HHVTGSDSLTTTGDSGPRNS ASSSSTSKLK RSRRTMARRL LPITLPARFK CLRTRSISSR TCSGRRTKAVSRRFQRTSSW SLSMDTSP SEQ ID NO: 4 LNPPSSPTPP RVSAKKASSR LKRSSFSKTKLEQATDPLRD QLPEPCLMTV RCVQQLAELQ SRADKVPMEW VMPRVIGIAI PPGLRATSRPPAPEPGSCPP TTTTSTSDSE RACSPTPTTD SPPPGDTLTS TASTATSHHV TGSDSSTTTGACDPKPCGSK SSTSRSRRSR RRTARQRWLI TLPARFRSLR TRRTNCRT SEQ ID NO: 5TTTFQKERRL GPKRTPSLPP RQTPKLDPAD PSSCKSQPNQ PQVWELIQCL REVAAHWATITKVPMEWAMP REIGIAIPRG WGTESSPSPP EPGCCPATTT TSTERSKAAP STEATPTPTLDTAPPGGTLT LTASTATGAP ETGKDSSTTT GASDPGPSES KSSTFKSKRS RCRTPPPPSPTTSPPPSKCL RTTTTSCPTS SATGPRDACR PSLRRSLRCR STVTRR SEQ ID NO: 6SSRHKSQTPP RALARQASSP LKRDSILVRL ATQSQSPTHN LSENLQQPPL LWDLLQWLQAVAHQWQTITK APTEWVMPQE IGIAIPHGWA TESSPPAPEH GPCPPITTTS TSKSPVLQRGPATTTTTSAT APPGGILIST DSTAISHHVT GSDSSTTIGD SGPRDSTSSS STSKSRRSRRMMASRPSLIT LPARFKSSRT RSTSCRTSSA LRTRAASLRS RRTCS SEQ ID NO: 7SRHLSVPPTP PRASARKASS PPERDSISVR LATQSQSPTL NLSENLQQRP LVWDLVQWLQAVAHQWQTIT KVPTEWVMPQ EIGIAIPHGW ATESLPPAPE PGPCPPTTTT STSKSPVKLQVVPTTTPTSA TAPPGGILTL TDSTATSHHV TGSDSSTTTG DSGPRSCGSS SSTSRSRRSRRMTALRPSLI TLPARFRYSR TRNTSCRTSS ALRTRAACLR SRRTSS SEQ ID NO: 8SHHPSVLQTP LRASARKANS PPEKDSILVR LATQSQFQTL NLSENLQQRP LVWDLIQWLQAVAHQWQTIT KAPTEWVVPR EIGIAIPHGW ATESSPPAPE PGPCPPTTTT STSKSPTGHREEPPTTTPTS ATAPPGGILT LTDSTATFHH VTGSDSSTTT GDSGPRDSAS SSSTSRSRRSRRMKAPRPSP ITSPAPSRCL RTRSTSCRTF SALPTRAACL RSRRTCS SEQ ID NO: 9SSLLRNRTPP RVLANRVHSP LKRDSISVRL ATQSQSQTLN QSENLPQPPQ VWDLLQWLQVVAHQWQTITK VPMEWVVPRE IGIAIPNGWG TESSPPAPEP GPCPPTTITS TSKSPTAHLEDLQMTTPTSA TAPPGGILTS TDSTATSHHV TGSDSSTTTG DSGLSDSTSS SSTFRSKRLRTTMESRPSPI TLPARSRSSR TQTISSRTCS GRLTRAASRR SQRTFS SEQ ID NO: 10TLGRLASQSQ SPTLNQSENH QQAPLVWDLV QWLQAVALQW QTITKAPTEW VVPQEIGIAIPHGWATESSP PAPEPGPCPP TTTTSTSKSP TGHREEAPTT TPTSATAPPG GILTSTDSTATSHHVTGSDS STTTGDSGQK DSASSSSTSR SRRSRRMKAP RPSPITLPAR FRYLRTRNTSCRTSSAPRTR AACLRSRRMS S SEQ ID NO: 11 SHHKSPTPPR ASAKKANNQP ERGSTLKRTLEPETDPLKDQ IPAPCLQTLK CVQHRAEMLS MRDKVPMEWV MPRVIGIAIP PGLRARSQQPRPEPGSCPPT TTTCTCVSEQ HQAATPTTDS PPPGDILTST DSTVTSHHVT GKDSSTTTGDYDQKPCALKS SISKLRRSQR RTARLRSLIT LPARFRYLRT RRMSSRT SEQ ID NO: 12KRLQIGRPTR TLGRPRPRKS KKTANQPTLL EGHSTLKTLE QETDPLRDHL PEKCLMMLRCVRRQAEMLSR RDKVPMEWVM PPVIGIAIPP GQRAESPPPA PEPGSYPRTT TTCTCESEQRPTATPTTDSP PPGDTLTLTA STATFPHATG SDSSTTTGDS GRNRCVLKSS TYRSRRSRRQTARLRSLITL PARFRSLRIR RMNSHT SEQ ID NO: 13 SRVLKSQTPR AELARKANSLPERDSTLTTN LEPETGLPQK DHLPELCLLR LKCVQQLAEM VAMRDKVPRE WVMPPVIGIAIPLGQRATSP PPQPAPGSCR PTTTTCTCGS ARATPATPST DSPPPGDTLT LTASTATSRQETGKGSSTTT GDCAPKACKS ASSTSKLRRS RRLTGRRPYP TTSPARSRSL RTARTSSRT SEQ IDNO: 14 VKPSSRPKRG FSNPLVWWKT QRRLRPETSG KAKTNLVCPT LLHRLPRKTR SLARKDLPAGQKIRAKAPLP TLEQQHPPLV WDHLSWLKEV AAQWAMQARV PMEWAIPPEI GIAIPNGWKTESSLEPPEPG SCPATTTTCT NESKDPAEAT TTTNSLDSAP PGDTLTTIDS TATFPRETGNDSSTTTGASV PKRCALDSLT SRLKRSRSKT STPPSATTSP VRSRSLRTRT TNCRTSSDRLPKAPSRRSQR ISTRSRSTGT AR SEQ ID NO: 15 ILVRLATQSQ SQTLNHSDNL PQPPLVWDLLQWLQAVAHQW QTITRVPMEW VIPQEIGIAI PNGWATESSP PAPAPGPCPP TTITSTSKSPANQEPPTTTT TLATAPPGGI LTSTDSTATF HHVTGKDSST TTGDSDPRDS TSSSLTFKSKRSRRMTVRRR LPITLPARFR CLLTPSTSSR TSSARRIRDA SRRSQQTSSW SHSMDTSP SEQ IDNO: 16 TRRTVSSLPL QRRPKLEALP PPAIWDLVRW LEAVARQSTT ARMVPMEWAM PREIGIAIPHGWTTVSSPEP LGPGICQPTT TTSTNDSTER PPETKATSDS APPGDTLTST ASTVISPLETGKDSSTITGD SDQRAYGSKS LTFKLKKSRR KTQRRSSPIT LPARFRYLRT RSTSSRT SEQ IDNO: 17 LNNPTTRPGP GRSVPNASTT FSRKRRRPRP SKAKPLLKRA KTPEKEPLPT LDQAPPLVWDHLSWLKEVAV QWAMQAKVPT EWAIPREIGI AIPNGWTTES LPEPLEPGSC PATTTTCTSGSKDREEPTPT INSLDSAPPG GTLTTTDSTA TSPPETGNDS STTTGASDPK RCALDSLTSRLKKSLSKTPT PPSPTTSPAR SKSLRTRTTS CRTSSDRLQR APSRRSQRIS TRSRSMVTAR SEQ IDNO: 18 TTTFQKERRL GPKRTPSLPP RQTPKLDPAD PSSCKSQHNQ PQVWELIQCL REVAAHWATITKVPMEWAMP REIGIAIPRG WGTESSPSPP APGCCPATTT TSTERSKAAP STEATPTPTLDTAPPGGTLT LTASTATGAP ETGKDSSTTI GASDPGLSES KSSTSKSKRS RCRTPPPPSPTTSPPPSKCL RTTTTNSRTS SATGPRDACR PSPRRSLRCR STATRR SEQ ID NO: 19ASRSRSWLLQ SSVHTRPRKP QRTRRVSRDR IPGRRPRRGS SSPISLDLQQ TYLHPHNSPSLPQGFPVWFL VRCLQEEALQ WTMLNKVPTE WAMPREIGIA IPNGWATEFS PDPPGPGCCPATTTTCTSRS QTPPACTASP GADTLATAPP GGTSTSIAST ATSRPETGSA SSITTGASDPRDCESNSSTS RSRRSRLLIR RPRSPTTSRA RSRSSQTTST SCRTSAATPP RDACRRSPRTSSRCRSTATR R SEQ ID NO: 20 KTEEPPRRAP NLWQHLKWQR EEAELWATLQ GVPMEWVMPREIGIAIPNGW ETQSSQRPPE PGSCQATTTT STKQLPVEPL KMQMSSMQDT VPPGGTLISTASTATSPLET GRDLSTTIGE SDPNLLNSRS SMSKSKKSQR RIKQRPLQTI SPQRFKSLRMMSINSRMSWA RLRKAPCRRS RRMSMPCRST GTAQCTPTRM EHGSMTVVHS TA SEQ ID NO: 21KSLNYLKKTL LHPVIVEEKQ VQLPPKAPNL WQHLTWQREE AELWATLQGV PMEWVMPQEIGIAIPNGWET QSLPRLQEPG SCQATTTTST KPSQAEQTQT QIPNMLDTAP PGGTLISTDSTAISLQETGR DSSTTIGGLD RKHSNSRYSM CKLKKSRRKT RQRLLLTTLP LQSRYSRIMNTSCPMFWARP RRGRCHRSPQ MCMPCPSTAT AQCTPTRVEL DSMTEVPSIA SEQ ID NO: 22TNTILKLKRP NKACRYQLHL KAEKKKLHRH NLEGAQQVPI LAAHLSWLQE EAVRWQTITRAPREWVIPQV IGIAIPSGWE TTSLQSQPEL GCSPLTGIIS TGLSTLTAPQ VRVLMQPMQDTRLPGGTLTS IDSIATSPPE TGKDSSTTTQ ASGRKDSKSK SLTSKSKKLQ HKIQRKQLPTISPAPYRSLR TRTTTYHMY SEQ ID NO: 143 LNNPTTRPGP GRSVPNASTT FSRKRRRPRPSKAKPLLKRA KTPEKEPLPT LDQAPPLVWD HLSWLKEVAV QWAMQAKVPT EWAIPREIGIAIPNGWTTES LPEPLEPGSC PATTTTCTSG SKDREEPTPT INSLDSAPPG GTLTTTDSTATSPPETGNDS STTTGASDPK RCALDSLTSR LKKSLSKTPT PPSPTTSPAR SKSLRTRTTSCRTSSDRLQR APSRRSQRIS TRSRSMVTAR

EXAMPLES

The following examples exemplify the invention for AAV, especially forAAV2. Due to the general similarities within the structures of theadeno-associated viruses and other parvoviruses the invention can beeasily transferred to other parvoviruses encoding 3 viral capsidproteins.

1. General Methods 1.1. Production of AAV (Like Particles) in InsectCells

For production of AAV particles in Sf9 cells (cultivated in Graces (JHRBioscience, USA)/10% FCS) cells were transfected with the vector plasmidpVL_VP1_MOD4, pVL_VP2 or pVL_VP3, derivates of the pVL1393 PolyhedrinPromoter-Based Baculovirus Transfer Vector (BD Bioscience, San Jose,Calif., USA) harboring a modified AAV VP1 open reading frame. (Cloningof pVL_VP1_MOD4, pVL_VP2 and pVL_VP3 is described in example 9)

Transfection was performed using the BaculoGold™ Transfection Kitaccording to manufacturer's manual (BD Bioscience, San Jose, Calif.,USA). Following transfection cells were incubated at 27° C. 5 days aftertransfection the supernatant was used for single clone separation via anend point dilution assay (EPDA). For that purpose Sf9 cells werecultivated in well plates (2×10⁴ cells/well) and infected with serialdilutions of the transfection supernatant. 7 days after incubation at27° C. the supernatant was transferred into a new 96 well plate (masterplate) and stored at 2-8° C. The cells of the EPDA are lysed with sodiumhydroxide, neutralized with sodium acetate and treated with ProteinaseK. Following an Immune detection with the DIG-DNA wash and Block BufferKit (Roche, Mannheim, Germany) single clones could be detected.

To amplify single clones the according well from the master plate wasused to infect Sf9 cells. Amplification of the recombinant Baculoviruswas performed through several passages. Each passage was incubated for 3days at 27° C. prior of use of the supernatant to infect cells for thenext passage. In the first passage 1.2×10⁵ Sf9 cells (12 well plates)were infected with 50 μl of the supernatant out of the according well ofthe master plate. Supernatant was used to infect 2×10⁶ Sf9 (T25 Flask)(passage 1B). For passage 2,2×10⁷ Sf9 (T175 Flasks) were infected with 1ml supernatant from passage 1B.

The virus titer of supernatant of passage 2 (P2) was analyzed via an endpoint dilution assay.

To produce AAV 1×10⁶/well Sf9 (6 well plates) were infected withsupernatant of P2 with a multiplicity of infection (MOI) of 1. Cultureswere incubated at 27° C. for 2-3 days. Cells were harvested anddisrupted by a freeze and thaw process and analyzed for AAV production.AAV2 titer was analyzed using a commercially available AAV2 titrationELISA kit (Progen, Heidelberg, Germany) according to the manufacturer'smanual.

1.2. Production of AAV (Like Particles) in Mammalian Cells 1.2.1.Plasmids

Ad Helper Plasmid

An Ad helper plasmid encoding adenoviral proteins E2, E4 and VAI-VAIIwas used for AAV manufacturing in 293 or 293-T cells. The helper plasmidpUCAdE2/E4-VAI-VAII was constructed by subcloning the BamHI restrictionfragment encoding the adenovirus (Ad) E2 and E4-ORF6 from pAdEasy-1(Stratagene, La Jolla, USA) into the BamHI site of pUC19 (Fermentas, St.Leon-Rot, Germany). The resulting plasmid is referred to as pUCAdE2/E4.The VAI-VAII fragment from pAdVAntage™ (Promega, Mannheim, Germany) wasamplified by PCR using the primers

XbaI-VAI-780-3′: (SEQ ID NO: 59) 5′-TCT AGA GGG CAC TCT TCC GTG GTC TGGTGG-3′, and XbaI-VAII-1200-5′: (SEQ ID NO: 60) 5′-TCT AGA GCA AAA AAGGGG CTC GTC CCT GTT TCC-3′

cloned into pTOPO (Invitrogen, Carlsbad, USA) and then subcloned intothe XbaI site of pUCAdE2/E4. This plasmid was named pUCAdV.

AAV Encoding Plasmids

The construction of pUCAV2 is described in detail in U.S. Pat. No.6,846,665. Plasmid pTAV2.0 is described in (Heilbronn et al., 1990),pVP3 is described in (Warrington et al., 2004). Further AAV viralprotein encoding plasmids are described within the respective examples.

1.2.2. Transfection for Large Scale Virus Production

293-T cells (ATCC, Manassas, USA) (7.5×10⁶/dish) were seeded in 15 cmdishes (i.e. dish with a diameter of 15 cm) 24 h prior to transfection(cultivated in DMEM/10% FCS). Cells were transfected by calciumphosphate precipitation as described in US 2004/0053410.

In case of AAV promoter p40 dependent transcription a co-transfectionwith an adenoviral helper plasmid was performed. For co-transfection ofthe AAV encoding plasmid and pUCAdV a molar ratio of the plasmids of 1:1was chosen. For transfection of one culture plate with 293-T cells thecalcium phosphate transfection protocol was used as described above, 12μg AAV Cap encoding plasmid (pUCAV2, pTAV2.0, and pVP3, respectively)and 24 μg pUCAdV were used. In case of p40 independent transcriptioncells were transfected with the respective AAV VP1, VP2 and/or VP3encoding plasmid. For transfection of one culture plate of 293-T cellsthe calcium phosphate transfection protocol was used as disclosed in US2004/0053410, 36 μg total DNA were mixed in 875 μl 270 mM CaCl₂. Inbrief, 875 μl 2×BBS (50 mM BES (N,N-Bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid) (pH 6.95), 280 mM NaCl and 1.5 mM Na₂HPO₄) was added tothe mixture and the resulting solution was carefully mixed by pipetting.The solution was incubated for 20 min at room temperature (RT) and thenadded drop-wise to the cell culture plate. After 18 h incubation ofcells in a humidified atmosphere at 35° C. and 3% CO₂, medium waschanged into a serum free DMEM (Invitrogen Carlsbad, USA) and cells werecultivated for an additional 3 d at 37° C., 5% CO₂ in a humidifiedatmosphere.

293-T cells were harvested with a cell lifter, transferred into 50 mlplastic tubes (Falcon) and centrifuged at 3000 g, 4° C. for 10 min. Thecell pellet was resuspended in 0.5 ml lysis buffer (150 mM NaCl, 50 mMTris, pH 8.5) per 15 cm dish and objected to three rounds of freeze andthaw cycles (liquid nitrogen/37° C.). The cell lysate was cleared by twocentrifugation steps (3700 g, 4° C., 20 min) and the AAV-containingsupernatant was used for further purification. Alternatively the wholedishes were objected to freeze and thaw cycles (−50° C./RT). Theremaining supernatant was collected and further purified as described in1.3.

1.2.3. Small Scale Transfection and Preparation of Virus Supernatants

Cells (5×10⁵/dish) were seeded in 6 cm dishes 24 h prior totransfection. 293-T cells were transfected by calcium phosphateprecipitation as described in US 2004/0053410. For HeLa and COS-1 cellstransfections were performed using Lipofectamine 2000 (Invitrogen,Carlsbad, USA) according to the manufacturer's manual. In case ofpromoter p40 dependent transcription of the cap gene (pTAV2.0, derivatesthereof, and pVP3) cells were infected with adenovirus type 5 (Ad5)(MOI=10). After additional incubation for 24-48 h, cells were harvestedin the medium and lysed by three freeze-thaw cycles (−80° C. and 37°C.). Lysates were incubated at 56° C. for 30 min to inactivate Ad5. Celldebris was removed by centrifugation at 10000 g for 5 min.

1.2.4. Cell Culture

HeLa and 293-T cells were maintained at 37° C. and 5% CO₂ in Dulbecco'smodified Eagle's medium (DMEM) supplemented with 10% heat-inactivatedfetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mML-glutamine.

1.3. Purification 1.3.1 Tangential Cross Flow Filtration (TFF) andBenzonase Treatment

After harvest the cleared cell culture medium was further concentratedusing a Tangential Cross Flow Filtration Unit (Sartoflow Slice 200Benchtop Crossflow System, Sartorius Biotech GmbH, Gottingen, Germany)using a 100 kDa cut off membrane (SARTOCON Slice 200). The resulting TFFconcentrate was pooled with the supernatant (obtained as described in1.2) and immediately treated with 100 U/ml benzonase (Merck, Darmstadt,Germany) at 37° C. for 2 h. After benzonase treatment the cell lysatewas cleared by centrifugation at 3700 g, 4° C. for 20 min. Clearedsupernatant was purified using size exclusion chromatography (ÅKTAexplorer system, GE Healthcare, Munich, Germany).

1.3.2 Size Exclusion Chromatography (SEC)

Cleared supernatant was separated through a Superdex 200 (prep grade)packed XK 50 chromatography column (250 mm in height and 50 mm indiameter; GE Healthcare, Munich, Germany). SEC fractions (5 ml each)were collected and the capsid titer was determined using the AAV2capsid-specific A20 ELISA (Progen, Heidelberg, Germany, Cat. No: PRATV).SEC fractions containing AAV2 particles were pooled and further purifiedusing iodixanol- or sucrose-density ultracentrifugation.

(i) Purification of AAV Particles by Density Gradient CentrifugationUsing Iodixanol

The virus-containing SEC pool was transferred to Qicksealultracentrifugation tubes (26×77 mm, Beckman Coulter, Marseille,France). Iodixanol solutions (purchased from Sigma, Deisenhofen,Germany) of different concentrations were layered beneath the viruscontaining lysate. By this an Iodixanol gradient was created composed of6 ml 60% on the bottom, 5 ml 40%, 6 ml 25% and 9 ml 15% Iodixanol withthe virus solution on top. The gradient was spun in an ultracentrifugeat 416000 g for 1 h at 18° C. The 40% phase containing the AAV particleswas then extracted with a canula by puncturing the tube underneath the40% phase and allowing the solution to drip into a collecting tube untilthe 25% phase was reached.

(ii) Sucrose Density Gradient Analysis

1.5×10⁶ cells were seeded in 10 cm dishes 24 h prior to transfection.They were harvested 48 h post transfection and lysed in 300 μl PBS-MK(phosphate-buffered saline: 18.4 mM Na₂HPO₄, 10.9 mM KH₂PO₄, 125 mM NaClsupplemented with 1 mM MgCl₂, 2.5 mM KCl) by five freeze-thaw cycles(−80° C. and 37° C.). After treatment with 50 U/ml Benzonase (Sigma,Deisenhofen, Germany) for 30 min at 37° C. and centrifugation at 3700 gfor 20 min the supernatant was loaded onto a 11 ml 5-30% or 10-30%sucrose gradient (sucrose in PBS-MK, 10 mM EDTA, containing one tabletof complete mini EDTA free protease inhibitor (Roche, Mannheim,Germany)) in polyallomer centrifuge tubes (14 by 89 mm; Beckman Coulter,Marseille, France). After centrifugation at 160000 g for 2 h at 4° C.(SW41 rotor; Beckman), 500 μl fractions were collected from the bottomof the tubes. As reference empty AAV2 capsids (60 S) were analyzed in aseparate gradient. For immuno dot blot assay 50 μl of heat denatured(99° C. for 10 min) or non denatured aliquots of the fractions weretransferred to Protran nitrocellulose membranes (Schleicher & Schuell,Dassel, Germany) using a vacuum blotter. Membranes were blocked for 1 hin PBS containing 10% skim milk powder and then incubated for 1 h withmonoclonal antibodies B1 (Progen, Heidelberg, Germany, Cat. No: 65158)to detect denatured capsid proteins or A20 to detect non denaturedcapsids. Antibodies B1 and A20 were applied in 1:10 dilutions. Membraneswere washed several times with PBS and incubated for 1 h with aperoxidase-coupled goat anti-mouse antibody (1:5000 dilution) (Dianova,Hamburg, Germany). Then, membranes were washed again and the antibodyreaction was visualized using an enhanced chemiluminescence detectionkit (Amersham, Braunschweig, Germany). For Western blot analysis 15 μlper fraction were processed for SDS-PAGE and then probed with monoclonalantibodies A69 (Progen, Heidelberg, Germany, Cat. No: 65157) or B1.

(iii) Purification of AAV Particles by ChromatographyPurification of Empty wtVP3# and Modified AAVLPs*

Indices # and * refer to slight differences in the purification protocolbetween wtAAV # and modified AAVLPs*. Buffer ingredients are markedcorrespondingly.

Cation Exchange Chromatography (ÅKTA Explorer System)

Total lysate containing empty wtVP3# and modified AAVLPs* was obtainedby performing three freeze thaw cycles (−54° C./37° C.). Total lysatewas cleared by centrifugation at 4100 rpm, 4° C., min (MULTIFUGE L-R;Heraeus, Hanau, Germany). The pH of the resulting cleared supernatantwas adjusted to 6. In addition, the conductivity of salt was reduced toapproximately 10 mS/cm by adding sterile water.

A Fractogel EMD SO₃ ⁻ (M) chromatography column (100 mm in height; 15 mmin diameter, XK16, GE Healthcare, München, Germany) was packed andequilibrated using 5 CV running buffer consisting of 80 mM NaCl, 2%sucrose, 50 mM HEPES (pH 6.0), 2.5 mM MgCl₂.

After equilibration, cleared supernatant was separated through theFractogel EMD SO₃ ⁻ (M) packed chromatography column (flow rate 10ml/min). After separation, column was washed using 5 CV running buffermentioned above. Bound particles (wtVP3 or modified AAVLPs) wereeffectively eluted at a sodium chloride concentration of 350 mM (peak1=45 ml).

Buffer Exchange (ÅKTA Explorer System)

To adjust the pH and the salt concentration of the eluted proteins(peak 1) for successive anion exchange chromatography, buffer exchangewas performed using a Sephadex G25 packed chromatography column (500 mmin height; 15 mm in diameter, XK26, GE Healthcare, München, Germany)(flow rate 10 ml/min). After column equilibration using 3 CV SOURCE 15Qrunning buffer consisting of 25 mM Tris (pH 8.2), 150 mM NaCl#/100 mMNaCl*, 2.5 mM MgCl₂ peak 1 was separated through the column. Proteinfraction (=120 ml) was collected.

Anion Exchange Chromatography (ÅKTA Explorer System)

A SOURCE 15Q chromatography column (80 mm in height; 15 mm in diameter,XK16, GE Healthcare, München, Germany) was equilibrated using 5 CVSOURCE 15Q running buffer consisting of 25 mM Tris (pH 8.2), 150 mMNaCl#/100 mM NaCl*, 2.5 mM MgCl₂. After equilibration, the proteinfraction obtained after buffer exchange (appr. 120 ml) was loaded andseparated through the chromatography column (flow rate 10 ml/min).Flow-through containing 90% of the particles (appr. 120 ml) wascollected.

Particle Concentration Using Centrifugal Filter Devices

Flow-through containing wtVP3# or modified AAVLPs* was concentratedusing Centricon Plus-(cut off 100 kDa) centrifugal filter devices(Millipore). Concentration was carried out using a swinging-bucket rotor(MULIFUGE L-R; Heraeus, Hanau, Germany) at 3500 g, 20° C. for 15 min.Resulting concentrate (appr. 45 ml) was immediately separated through asize exclusion chromatography.

Size Exclusion Chromatography (ÅKTA Explorer System)

A Superdex 200 (prep grade) chromatography column (500 mm in height; 50mm in diameter, XK50, GE Healthcare, München, Germany) was packed andequilibrated using 2 CV running buffer consisting of 200 mM NaCl, 2%sucrose, 50 mM HEPES (pH 6.0), 2.5 mM MgCl₂. The concentrate mentionedabove (appr. 45 ml) was separated through the column (fow-rate 10ml/min). Particles eluted first (SEC fraction no. 1-13; each 5 ml). SECfractions with a particle purity of greater than 95% were pooled,sterile filtered (0.2 μm) (Minisart; Sartoriusstedim) and stored at −84°C.

1.4. Analysis of Protein Expression by Western Blot

Identical portions of harvested cells or identical amounts of purifiedparticles were processed for SDS-PAGE. Protein expression was analyzedby Western blot assay using monoclonal antibodies A69, B1 (Progen,Heidelberg, Germany), anti-AU1 (Covance, Emeryville, USA), anti-GFP(clone B-2; Santa Cruz Biotechnology, Santa Cruz, USA) or polyclonalantibody anti-AAP (see 1.7.) as described previously (Wistuba et al.,1995). Variations of the protocols are indicated within the descriptionof the respective examples.

1.5. Titer Analysis

Capsid titers were determined using a commercially available AAV2titration ELISA kit (Progen, Heidelberg, Germany Cat. No: PRATV) or therespective AAV1 titration ELISA kit (Progen, Heidelberg, Germany Cat.No: PRAAV1) according to the manufacturer's manual.

1.6. Immunofluorescence Analysis

For immunofluorescence analysis HeLa cells were cultivated for 24 h oncoverslips, transfected and in case of promoter p40 dependenttranscription of the cap gene (pTAV2.0 and pVP3) infected with Ad5(MOI=4). After 20-48 h cells were fixed with 100% methanol (10 min, −20°C.) and washed with PBS (phosphate-buffered saline: 18.4 mM Na₂HPO₄,10.9 mM KH₂PO₄, 125 mM NaCl). Incubation with primary antibodies wasperformed for 1 h at RT or over night at 4° C. As primary antibodieshybridoma supernatants A20 or A69 were used to detect assembled capsidsor VP2 respectively. A20 and A69 were used undiluted (Progen,Heidelberg, Germany). For detection of unassembled capsids a rabbitpolyclonal serum was used in a 1:500 dilution to label all three free VPproteins. Coverslips were washed three times with PBS and thereafterincubated with appropriate secondary antibodies (Cy 3 labeled goat antimouse in 1:400 dilution or FITC labeled goat anti rabbit 1:150 purchasedfrom Dianova, Hamburg, Germany or Molecular Probes, Leiden, TheNetherlands) for 1 h at RT. Coverslips were washed again, dipped into100% ethanol and embedded in Permafluor mounting medium (BeckmanCoulter, Marseille, France). Confocal images (0.3 μm sections) wereobtained with a Leica TCS SP2 laser scanning microscope and furtherprocessed using Adobe Photoshop CS software. Variations of the protocolsare indicated within the description of the respective examples.

To visualize GFP expression, cells were fixed with 2% paraformaldehydefor 15 min, quenched twice with 50 mM NH₄Cl for 5 min, and permeabilizedwith 0.2% Triton X-100 for 10 min.

1.7. Preparation of Polyclonal Antibody

The polyclonal AAP antiserum (anti-AAP) was generated by immunization ofa guinea pig with a peptide comprising the sequence GKDSSTTTGDSDPRDSTS(SEQ ID NO: 61) conjugated to KLH (Keyhole Limpet Hemocyanin) followingstandard procedures.

1.8. Negative Staining of Virus Particles for Electron Microscopy

For electron microscopy according to (Grimm et al., 1999, Grimm andKleinschmidt, 1999, Mittereder et al., 1996), negative staining of virusparticles was performed as described in detail below.

Five μl of sample (about 5×10¹⁰ virus particles) were applied onto thefreshly air-glow discharged carbon coated side of a grid and incubatedfor 2 min. Excess solution was removed by blotting the edge of the gridonto Whatman filter paper. To avoid salt precipitates, the grid waswashed with 3 drops of water followed by four drops of 2% (w/v) uranylacetate solution. The last droplet of staining solution was allowed tosit on the grid for 5 min before blotting and air drying. Electronmicrographs were taken with a Morgagni 268D FEI microscope at 100 kV.

2. Analysis of VLP Formation by N-Terminal Deletion Analysis of VP2

Our as well as previous studies (compare above) reported a lack ofcapsid assembly when VP3 is expressed from constructs comprising the cdsof VP3 alone. Since expression of VP3 is not sufficient for VLPformation, we tried to identify further sequences which could overcomethis defect. In this experiment we checked whether a sequence upstreamof the VP3 cds was necessary for VLP formation. If yes, the sequenceshould be characterized.

2.1. Cloning of Deletion Mutants

Plasmids pTAV2.0 (Heilbronn et al., 1990), pVP3 (Warrington et al.,2004), pCMV-VP (Wistuba et al., 1997) and pKEX-VP3 (Ruffing et al.,1992) have been described previously. The deletion mutantspCMV-VP3/1882, pCMV-VP3/2193, pCMV-VP3/2596, pCMV-VP3/2611,pCMV-VP3/2696, pCMV-VP3/2765 and pCMV-VP3/2809 were cloned from plasmidpVP3. Numbers behind the name of the pCMV-VP3 plasmid indicate thenucleotide position in the AAV2 genome according to Ruffing et al.(1994). Constructs are schematically shown in FIGS. 5A and 5B.

For cloning of deletion mutants, the HindIII/BsiWI fragment of pVP3(with mutated VP1 and VP2 translation start codons) was subcloned intothe HindIII/BsiWI backbone of pCMV-VP resulting in the constructpCMV-VP3/1882 (FIGS. 5A-5D). Constructs pCMV-VP3/2193 and pCMV-VP3/2596were generated by subcloning of the DraI/BsiWI or the EcoNI(blunted)/BsiWI fragment from pVP3 into the HindIII (blunted)/BsiWIbackbone of pCMV-VP (EcoNI and HindIII sites were blunted by digestionof the single stranded overhang) (the position of the differentrestriction sites used for cloning relative to the genomic sequence isshown in FIG. 4). For further deletions pVP3 was used as a template forsite-directed mutagenesis reactions. Mutagenesis was performed using aQuickChange site-directed mutagenesis kit (Stratagene, Amsterdam, TheNetherlands) according to the manufacturer's manual. For each mutation,two complementary PCR primers were designed to generate a new HindIIIrestriction site at the designated area. Primer sequences:

(SEQ ID NO: 62) 5′-CCTCTGGTCTGGGAACTAAGCTTATGGCTACAGGCAGTGGCG-3′ (SEQ IDNO: 63) 5′-CGCCACTGCCTGTAGCCATAAGCTTAGTTCCCAGACCAGAGG-3′

HindIII/BsiWI fragments from mutated plasmids were then subcloned intothe HindIII/BsiWI backbone of pCMV-VP resulting in constructspCMV-VP3/2611, pCMV-VP3/2696, pCMV-VP3/2765 and pCMV-VP3/2809 (FIGS. 5Aand 5B).

2.2. Analyses of Constructs by Western Blot and ELISA

For analysis of protein expression identical portions of harvested cellswere processed for SDS-PAGE.

As shown in FIG. 5C, transfection of 293-T cells with all constructslisted in FIGS. 5A and 5B except pTAV2.0 (wt AAV) and pCMV-VP resultedin expression of only VP3 when analyzed by Western blotting usingantibody B1 which reacts with all three capsid proteins. In contrastcells transfected with pTAV2.0 (wt AAV) or pCMV-VP, a plasmid in whichthe corresponding translation start sites were not mutated, VP1 and VP2were well detected in addition to VP3. Antibody B1 reacted with twopolypeptide bands migrating slower than VP3 e.g. for mutated plasmidspKEX-VP3, pCMV-VP3/2765 and pCMV-VP3/2809. At least for plasmidspKEX-VP3 and pCMV-VP3/2809 the corresponding polypeptides cannot containVP1 or VP2 amino acid sequences since the nucleotide sequences codingfor VP1 or VP2 were completely deleted. Moreover, VP1 and VP2 could notbe detected upon expression of all three mutant plasmids, using theantibody A69. Hence, the presence of VP1 and VP2 in these samples couldclearly be excluded. We concluded that the two polypeptide bandsmigrating slower than VP3 were a consequence of higher VP3 levels, whichwere not completely denatured.

When, however, extracts of cells transfected with pVP3 were probed withantibody A69 which detects only VP1 and VP2, thus omitting the reactionwith the abundant VP3, one could detect faint bands in the region of VP1and VP2 which were absent in extracts of cells transfected withpKEX-VP3. This result suggests that transfection of the pVP3 constructleads to the expression of small amounts of VP1 and VP2 or VP1- andVP2-like proteins. They are possibly translated from alternativetranslation initiation codons or by unscheduled initiation at themutated VP1 and VP2 translation initiation sites.

Antibody A69 revealed in all deletion mutants of pVP3 up topCMV-VP3/2696 one or several polypeptide band(s), only Western blotswith extracts of cells transfected with pCMV-VP3/2765 and pCMV-VP3/2809showed no reaction with A69 because the antibody epitope was alreadydeleted in these proteins.

Capsid assembly was confirmed by an antibody A20 based capsid ELISA(FIG. 5D). In contrast, expression of VP3 by pKEX-VP3 did not yielddetectable amounts of capsids (FIG. 5D), although the amount ofexpressed VP3 protein was even higher compared to pVP3 (FIG. 5C).

In agreement with our previous results, expression of VP3 alone bytransfecting pCMV-VP3/2809—which is equivalent to pKEX-VP3—did not leadto detectable capsid formation (FIG. 5D). The formation of capsids whichmight not react with the A20 ELISA was excluded by analysis of cellextracts on sucrose gradients followed by Western blotting with the B1antibody (data not shown). Interestingly, analyzing the capsid assemblyefficiency of the different deletion mutants it was detected that thecapsid assembly efficiency increased from one deletion mutant to thenext, before decreasing upon a certain extent of deletion. Peakefficiencies in capsid assembly were seen for mutants pCMV-VP3/2596 andpCMV-VP3/2611 (FIGS. 5A, 5B, and 5D).

2.3 Conclusion

This result shows a clear correlation between the presence ofN-terminally extended VP3 sequence (due to the presence of DNA sequenceupstream of the VP3 start codon) and capsid assembly. We identified aDNA sequence of about 44 nucleotides upstream of the VP3 cds that has tobe present in addition to the VP3 cds for VP3 VLP formation. This 44 ntconfers to construct pCMV-VP3/2765 which still is able to cause capsidassembly.

The presence of some more DNA sequence upstream of the 2765′ siteincreases efficiency of capsid assembly which is in line with ORF2starting at nucleotide position 2717 and the putative start of thefull-length AAP possibly located between nucleotide 2717 and 2765.

3. Sequence Fragment of the Cap Gene is Able to Induce Capsid Assemblyin Trans

In example 2, we identified some sequence upstream of the VP3 startcodon (comprised by fragment Z) that has to be present in addition tothe VP3 cds for particle formation. To prove the hypothesis that theproduct of fragment Z functions transiently and in trans, we testedwhether a capsid sequence fragment comprising the EcoNI/BsiWIrestriction fragment fused to the cds of GFP can rescue the capsidassembly deficiency of VP3.

3.1. Cloning of pVP2N-gfp for Trans-Complementation

For generation of construct pVP2N-gfp, EcoNI and BsiWI restriction siteswere introduced into the multiple cloning site of the vector pEGFP-N1(BD Biosciences, Erembodegem, Belgium). Afterwards the EcoNI/BsiWIfragment from pTAV2.0 (position of restriction sites is given in FIG. 4)was inserted downstream of a CMV promoter and upstream of the GFP cdsand its poly(A) signal. Expression of this fusion construct pVP2N-gfpresults in three transcripts VP2N-gfp, VP3N-gfp and GFP, depending onthe initiation of transcription at one of the three existing startcodons for VP2, VP3 or GFP as schematically shown in FIG. 6A.

A number of derivates containing e.g. codon modifications or stop codonsoriginated from pVP2N-gfp as schematically indicated in the respectivefigures. They always include the GFP cds and were named accordingly(with the addition −gfp). To simplify matters this appendix (−gfp) ismissing to names of the respective constructs in some figures (e.g.FIGS. 20, 22, 23).

3.2. Analysis of Functional Substitution in Trans

The following experiments were performed in HeLa cells. PlasmidspCMV-VP3/2809 and pVP2N-gfp were co-transfected in different molarratios and analyzed for gene expression and capsid assembly (FIGS.6A-6D). While Western blot analysis confirmed that the amount of VP3 wasthe same in cell extracts transfected in each molar ratio of the twoplasmids (detection with antibody B1, FIG. 6B upper part), VP2N-gfp(detection with antibody A69, FIG. 6B lower part) could only be detectedafter transfection in a 1:1 or 1:1/5 ratio, respectively. In the 1:1 or1:1/5 ratio, antibody anti-gfp (FIG. 6B, middle) additionally detectsall three transcripts resulting from expression of the fusion constructpVP2N-gfp as schematically shown in FIG. 6A, namely VP2N-gfp, VP3N-gfpand GFP. Due to the strong start codon of VP3 and corresponding to thein vivo situation the transcript of VP3N-gfp dominates. Surprisingly,capsid assembly could be observed by immunofluorescence up to apCMV-VP3/2809 to GFP-fusion-plasmid ratio of 1:1/50 (FIG. 6C).Quantification of capsid formation using the antibody A20 based capsidELISA showed that capsid formation of mutant pCMV-VP3/2809 supplementedwith pVP2N-gfp was similarly efficient as mutant pCMV-VP3/2696 where theN terminally extended VP3 was co-expressed (FIG. 6D).

3.3. Conclusion

This result shows that presence of an EcoNI-BsiWI restriction fragmentof the cap gene in trans rescues capsid assembly of constructsexpressing VP3 as only capsid protein. Since assembly could be detectedeven at a 50-fold reduced amount of pVP2N-gfp plasmid co-transfected, asubstoichiometric action of the helper factor for VP3 capsid assemblycan be assumed.

4. C-Terminally Truncated VP2 Proteins are Expressed in SubstoichometricAmounts and Become Incorporated into Capsids

Here it was investigated if the generated AAV like particles consist ofVP3 only. Empty particles were produced from plasmid pCMV-VP3/2696 or ina trans-complementation assay of cotransfection of pCMV-VP/2809 andpVP2N-GFP Particles were purified via sucrose cushion according toSteinbach et al. (1997) with modification described by Kronenberg et al.(2001) and with the modification that the 293 cells were transfectedwithout adenoviral infection and cells were harvested after 48 h.Incorporation of truncated VP2 protein was analyzed by Western blot(FIG. 7).

pVP2N-GFP could not be detected within maximal loading of 5×10¹¹particles. But transfecting pCMV-VP3/2696 an A69 signal was detectedwhich shows that a truncated VP2 is incorporated into the capsidssubstoichiometrically.

4.1. Result

In conclusion VP3 only particles are generated within the transsituation. In contrast in the cis situation a truncated VP2 isincorporated substochiometrically. From Western blot the signalintensity of VP1 from 2×10⁹ wt AAV particles is about the same as thesignal from 1×10¹¹ particles generated from pCMV-VP3/2696. This meansthe amount of truncated VP2 is about fold lower than the amount of VP1.Assuming a stoichiometric ratio of VP1:VP2:VP3 of 1:1:10 within a wtcapsid there is approximately 500-fold less truncated VP2 than VP3.Since one capsid is composed of 60 VP subunits also capsids must existthat are composed of VP3 only.

4.2. Conclusion

This result strengthens the conclusion that the truncated VP2 proteinitself is not required for the capsid itself.

5. Codon Modification of the Construct Used for Trans-Complementationcan Inhibit the Trans-Complementation Process

To investigate the nature of the trans-complementing agent of thefragment Z, the VP2N part (part between restriction sites EcoNI andBsiWI) within pVP2N-gfp was codon modified. That means the DNA sequencewas altered without changing the amino acid sequence of the first ORF.Codon modification was performed by GENEART (Regensburg, Germany).Codons were modified for codons preferentially used in mammalian cells.Sequence is shown in FIGS. 8A-8C. Identity of the DNA sequence ofpVP2N-gfp versus pVP2N-gfp codon modified (cm, pVP2Ncm-gfp) is 71% whileprotein identity is 100%.

Protein expression of pVP2Ncm-gfp was compared in Western blot analysis(FIG. 8D) and by immunoflourescence within transfected 293 cells (FIG.8E). The ability to rescue capsid formation of pCMV-VP3/2809 was testedin trans-complementation assays as described in example 3 (FIG. 8F).Plasmids were cotransfected in a molar ratio of 1:1.

Result and Conclusion

Western blot showed that the protein expression from the codon modifiedconstruct (pVP2Ncm-gfp) was even higher than protein expression from thenon-modified construct (pVP2N-gfp), not unexpected since the codonmodification was optimized for mammalian cells (FIG. 8D). Also thelocalization within the cells of the codon modified protein did notdiffer from the non-modified protein (FIG. 8E). Surprisingly thepVP2Ncm-gfp lost its ability to rescue capsid formation of pCMV-VP3/2809(FIG. 8F).

To exclude a negative effect of the large amounts of pVP2Ncm-gfp proteinon capsid assembly, we co-transfected the codon modified pVP2Ncm-gfpwith pCMV-VP3/2696. In this combination capsid assembly was normal,showing that the assembly activity was not suppressed by the high amountof pVP2Ncm-gfp (data not shown). Also expression of lower amounts ofpVP2Ncm-gfp by transfection of reduced amounts of plasmid pVP2Ncm-gfptogether with pCMV-VP3/2809 did not rescue capsid assembly (data notshown).

This result strengthens the hypothesis that no protein translated fromthe first ORF is responsible for the trans-complementing activity.

6. Insertion of Stop Codons into the Construct Used forTrans-Complementation does not Inhibit the Trans-Complementation Process

To further analyze the nature of the trans-complementing agent stopcodons were inserted within the EcoNI-BsiWI restriction fragment. Toinsert Stop codons point mutations were performed.

6.1. Insertion of Stop Codons into pVP2N-gfp

For construction of pVP2N/stopA-gfp (also named pVP2N/ORF1stopA-gfp),pVP2N/stopB-gfp (identical to pVP2N/ORF1 stopB-gfp), pVP2N/stopC-gfp(also named pVP2N/ORF1 stopC-gfp) and pVP2N/stopD-gfp (identical topVP2N/ORF1 stopD-gfp) site-directed mutagenesis reactions (QuickChangesite-directed mutagenesis kit, Stratagene) were performed using templatepVP2N-gfp and two complementary PCR primers which included the desiredsubstitutions. In each case the EcoNI/BsiWI fragment was then clonedinto the EcoNI/BsiWI backbone of pVP2N-gfp.

For generation of StopA cytosine at nucleotide position 2770 weresubstituted to thymine resulting in a tag stop codon. For generation ofStopB adenine at nucleotide position 2797 was substituted to thymineresulting in a tga stop codon. Stop C was generated by substitutingadenine at nucleotide position 2821 to thymine and thymine at position2823 to adenine, resulting in a tga stop codon. Stop D was created bysubstituting guanine at nucleotide position to thymine resulting in atga stop codon. Positions are according to Ruffing et al. (1994).

Primer pairs used for insertion of Stop codons at four different siteswithin the pVP2N-gfp StopA 5′-CCA GCC TCT CGG ATA GCC ACC AGC AGC C-3′(SEQ ID NO: 64) i-StopA 5′-GGC TGC TGG TGG CTA TCC GAG AGG CTG G-3′ (SEQID NO: 65) StopB 5′-GCC CCC TCT GGT CTG TGA ACT AAT ACG ATG GC-3′ (SEQID NO: 66) i-StopB 5′-GCC ATC GTA TTA GTT CAC AGA CCA GAG GGG GC-3′ (SEQID NO: 67) StopC 5′-CGA TGG CTA CAG GCT GAG GCG CAC CAA TGG C-3′ (SEQ IDNO: 68) i-StopC 5′-GCC ATT GGT GCG CCT CAG CCT GTA GCC ATC G-3′ (SEQ IDNO: 69) StopD 5′-GGA GTG GGT AAT TCC TCG TGA AAT TGG CAT TGC G-3′ (SEQID NO: 70) i-StopD 5′-CGC AAT GCC AAT TTC ACG AGG AAT TAC CCA CTC C-3′(SEQ ID NO: 71)

Schematic presentation of the inserted stop codons is depicted in FIG.9A. In pVP2N/stopA-gfp nucleotide c₂₇₇₀ has been mutated into t,therefore the cag-codon encoding glutamine is changed into tag (silentmutation in ORF2), in pVP2N/stopB-gfp nucleotide g₂₇₉₇ has been mutatedinto t, hence the gga-codon encoding glycine is changed into tga(Trp→Cys mutation in ORF2), in pVP2N/stopC-gfp nucleotide a₂₈₂₁ has beenmutated into t (silent in ORF2) and nucleotide t₂₈₂₃ has been mutatedinto a, therefore the agt-codon encoding serine is changed into tga(Val—Glu mutation in ORF2), and in pVP2N/stopD-gfp nucleotide g₂₈₇₈ hasbeen mutated into t, hence the gga-codon encoding glycine is changedinto tga (silent in ORF2). Positions are according to Ruffing et al.(1994). All substitutions do not disrupt ORF2. The resulting pVP2N-gfpstop constructs were used for trans-complementation of the constructpCMV-VP3/2809 as described in example 3. Plasmids pCMV-VP3/2809 and therespective pVP2N/stop-gfp construct were cotransfected in a molar ratioof 1:1.

Further protein expression of the Stop constructs was tested by Westernblot analysis using the A69 antibody.

6.2. Result and Conclusion

Western blot analysis confirmed that VP3 is expressed in all samples(detected by monoclonal antibody B1 in FIG. 9B). As expected Bluescriptvector (pBS) did not cause capsid assembly in the trans-complementationassay and therefore served as a negative control (FIG. 9C).Interestingly, although no protein expression was detected for thepVP2N/stop-gfp constructs in contrast to the pVP2N-gfp construct (FIG.9B), the insertion of stop codons did not influence thetrans-complementing activity of the EcoNI-BsiWI restriction fragment ofthe cap gene. VP3 particles could easily be assembled (FIG. 9C). Thereduction in capsid titers obtained with mutants pVP2N/stopB-gfp andpVP2N/stopC-gfp could be due to the nucleotide changes introduced bygenerating the respective mutations (stopB in ORF1 led to a Trp-Cysmutation in ORF2, stopC in ORF1 led to a Val-Glu mutation in ORF2).These experiments together show that the nucleic acid sequence of theEcoNI-BsiWI fragment is the basis for the capsid assembly helperactivity and not an expressed protein from the first ORF, since allmutants contain stop codons in the first ORF. Although the substitutionsresulting in stop codons in ORF 1 did not stop amino acid synthesis ofAAP from ORF2, differences in capsid titers indicated that thefunctionality of AAP was influenced.

7. The Postulated NLS is not Necessary for VLP Formation

While mutant pCMV-VP3/2696 formed high capsid levels, the slightlyshorter mutant pCMV-VP3/2765 assembled to clearly reduced amounts ofcapsids (FIG. 5D). This shorter mutant had lost a group of AA which hadbeen suggested to function as a NLS for AAV VP2 proteins (Hoque et al.,1999a) and showed reduced nuclear transport of the VP protein (FIG. 10)To test whether the postulated NLS is responsible for this difference,we substituted the respective sequence element by converting the RKRpeptide (AA 168-170) into AAA in the construct pCMV-VP3/2696 in order todestroy the proposed NLS activity by site directed mutagenesis accordingto standard procedures using two complementary PCR primers whichincluded the desired substitutions. Primers used for substitution of RKRby AAA:

BC3-ala forward: (SEQ ID NO: 72) 5′-GGC GGG CCA GCA GCC TGC AGC AGC AGCATT GAA TTT TGG TCA GAC TGG-3′ BC3-ala reverse: (SEQ ID NO: 73) 5′-CCAGTC TGA CCA AAA TTC AAT GCT GCT GCT GCA GGC TGC TGG CCC GCC-3′

Immunofluorescence of transfected HeLa cells with the A20 antibody (FIG.10) and the capsid ELISA (data not shown) showed that the VP protein ofmutant pCMV-VP3/2696RKR168-170AAA was as active in capsid assembly as wtAAV.

This supports the interpretation that the sequence element comprisingRKR168-170 does not act as a NLS in this context and might play adifferent role in capsid assembly.

8. Nuclear Localization (and N Terminal Extension) of VP Proteins is notSufficient for Capsid Assembly

It has been reported that fusion of an NLS derived from the SV40 large Tantigen to VP3 translocates VP3 into the nucleus and leads to capsidassembly (Hoque et al., 1999a). We repeated this experiment and observedefficient nuclear accumulation of VP3 protein, however, there was nocapsid assembly detectable with antibody A20 (FIGS. 11A, 11B and 15B).

Further, a heterologous N terminal extension upstream of VP3 (HSA) wastested to restore assembly competence to VP3.

Further several constructs were transfected in 293 cells to compareprotein expression and assembly efficiency.

8.1. Cloning of Constructs

pCI-VP, pCI-VP2 and pCI-VP3 were cloned by PCR amplification of therespective VP coding regions using primer with XhoI (5′-) and NotI (3′-)overhangs and subcloning of the XhoI-/NotI-digested PCR products intothe XhoI-/NotI-digested vector pCI (PROMEGA). In case of pCI-VP2, thestart codon for VP2 was changed from ACG to ATG at the same time

Cloning of the construct pCMV-NLS-VP3 was carried out by site-directedmutagenesis reaction using the construct pCMV-VP3/2809 as template andthe complementary PCR primers

(SEQ ID NO: 74) 5′-GGAAT TCGAT ATCAA GCTTG CCATG GCACC ACCAA AGAAG AAGCGAAAGG TTATG GCTAC AGGCA GTGG-3′ and (SEQ ID NO: 75) 5′-CCACT GCCTG TAGCCATAAC CTTTC GCTTC TTCTT TGGTG GTGCC ATGGC AAGCT TGATA TCGAA TTCC-3′.

Then the HindIII/BsiWI fragment was subcloned from the amplicon into theHindIII/BsiWI backbone of pCMV-VP3/2809. The cap gene product NLS-VP3contains the amino acid sequence of SV40 NLS MAPPKKKRKV at theN-terminus of VP3.

The construct pCMV-HSA-VP3 is also based on pCMV-VP3/2809 and contains anucleic acid sequence coding for amino acids 25-58 of human serumalbumin (HSA) directly upstream of the VP3 cds. Fragment

(SEQ ID NO: 76) 5′-GGTAC CAAGC TTACG GACGC CCACA AGAGC GAGGT GGCCC ACCGGTTCAA GGACC TGGGC GAGGA AAACT TCAAG GCCCT GGTGC TGATC GCCTT CGCCC AGTACCTGCA GCAGT GCAAG CTTGA GCTC-3′(with a HindIII restriction site at both ends) was obtained via genesynthesis (Geneart, Regensburg, Germany). After HindIII digestion of thecorresponding vector the resulting 111 bp fragment was subcloned intothe HindIII linearized pCMV-VP3/2809 backbone. Translation of VP3 isinitiated at a standard ATG start codon whereas translation of HSA-VP3(with 37 Aas elongation at VP3 N-terminus) is initiated at an ACG startcodon.

8.2. Analyses of Constructs by Immunofluorescence and Sucrose Gradient

We transfected HeLa cells with the different constructs: pCMV-NLS-VP3 orpCMV-VP3/2809 either alone or in a co-transfection with pVP2N-GFP.Further pCMV-HSA-VP3 was transfected. Expression of capsid proteins andformation of capsids was analyzed by immunofluorescence as describedabove using a polyclonal VP antiserum or the monoclonal A20 antibody.Further capsid formation was analyzed within following a sucrosegradient.

Results

Just as Hoque et al. (1999a) and comparable to the wildtype (wt) and theproteins expressed from the N-terminally truncated construct pCMV/2696,we could express VP3 from the construct pCMV-NLS-VP3 and observedefficient nuclear accumulation of VP3 protein. However, in contrast tothe wt and the N-terminally truncated construct pCMV/2696 we could notdetect capsid assembly using the antibody A20 (FIG. 11C).

As expected, expression of the VP3 protein with a prolonged N-terminusconsisting of 36 AA of human serum albumin (HSA-VP3), equivalent inlength to the VP3 N-terminal extension of mutant pCMV-VP3/2696 could bedetected by antibody staining (FIG. 11C). In comparison to theexpression product of pCMV-NLS-VP3 those of the mutant pCMV-HSA-VP3showed a much higher fraction of cytoplasmic staining. Again, we couldnot detect capsid assembly using the antibody A20 (FIG. 11C).

Co-transfection of pVP2N-gfp induced capsid assembly, readily detectableby antibody A20 (FIG. 11C).

Analysis of possible assembly products—not reacting with the A20antibody—by sucrose density gradient sedimentation showed very lowamounts of VP protein containing material (sedimenting over the wholerange of the gradient) which reacted with antibody B1 (FIG. 11B). Thisindicates the formation of incorrectly assembled or aggregated VPprotein in rather low, hardly detectable quantities.

8.3. Analyses of Constructs by Western Blot and ELISA

A set of different constructs was analyzed for gene expression inWestern Blot and in ELISA for capsid assembly (FIG. 15A):

-   -   pCI-VP2: The VP2 sequence of AAV2 was cloned into the multiple        cloning site of pCI (Promega, Mannheim, Germany). The VP2 start        codon ACG was changed into an ATG.    -   pCI-VP3: The wildtype VP3 sequence was cloned into pCI.    -   pCI-VP: The complete cap ORF was cloned into pCI. The start        codons of VP2 and VP3 were not mutated.    -   pCMV-NLS-VP3: (described in example 8 and by Hoque et al.        (1999a))    -   pCI-VP2mutACG: This is a modification of the pCI VP2: the VP2        start-codon is destroyed and replaced by a GAG codon    -   pCMV-VP3/2696 (described in example 2)

Results

Western Blot analysis showed similar capsid protein expression of thedifferent constructs with the expected size of the VP proteins (FIG.15C). The efficiency in capsid assembly however was quite different(FIG. 15B). Particle titer obtained with the construct cloned analogueto Hoque et al (pCMV-NLS-VP3) was below detection limit. That also meansthat the favorised constructs pCI-VP2mutACG or pCMV-VP3/2696 are morethan 3 log more efficient in VP3 particle formation efficiency whencompared to the Hoque construct pCMV-NLS-VP3. The construct pCI-VP2corresponds to pCMV-VP3/2611 except for a mutation of the minor ACGstart codon to an ATG in pCI-VP2 whereas the ACG codon is completelydeleted in pCMV-VP3/2611. Capsid formation efficiency of the pCI-VP2construct is strongly reduced (FIG. 15B). We did not analyze whether theparticles obtained from pCI-VP2 are mainly composed of VP2, VP3 or amixture of both proteins. FIG. 15C shows that VP3 is still expressedfrom this construct even though with significantly (about 10 fold) lowerefficiency compared to VP2. We hypothesize that the particles obtainedmainly consist of VP3. The low titer is explained by i) 10-fold reducedamounts of VP3 from pCI-VP2 compared to pCMV-VP3/2611. Furthermore, wespeculate that the ATG start codon in pCI-VP2 interferes with AAPexpression as the ATG probably dominates the non-canonical start codonof AAP. pCI-VP3 showed only low capsid formation efficiency as expected.Efficiency of particle assembly could partially be rescued byco-transfection of pCI-VP3 with pCI-VP2 (FIG. 15B) in a ratio of 10:1.However, the overall particle formation is still reduced by 1-2 logcompared to pCI-VP2mutACG or pCMV-VP3/2696 supporting our hypothesisthat the ATG start codon in the VP2 coding region of pCI-VP2 interfereswith AAP expression. Particle formation from pCI-VP is much lower whencompared to pCMV-VP (FIGS. 5A-5D). This is explained as follows: pCI-VPdiffers from pCMV-VP by lack of the splice donor site. Therefore, onlyone messenger RNA is transcribed from pCI-VP expressing mainly VP1,whereas two messenger RNAs are transcribed from pCMV-VP. The minortranscript mainly expresses VP1, whereas the major transcript encodesVP2 and VP3 in a ration of 1:8. Therefore, pCMV-VP expresses VP1:VP2:VP3in the expected ratio of 1:1:8, whereas VP2 and VP3 can hardly if at allbe detected with construct pCI-VP.

Conclusion

The results show that nuclear accumulation of VP3 alone is notsufficient for capsid assembly and that a heterologous N-terminalextension upstream of VP3 is not able to bring about assembly competenceto VP3.

Further our favored constructs pCI-VP2mutACG or pCMV-VP3/2696 lead tomore than 3 log higher VP3 particle titers when compared to the NLS-VP3fusion construct described by Hoque et al. (1999a). These experimentsalso demonstrate that VP3 N-terminal fusion constructs can assemble intoVLPs. Therefore I-203 is a suitable insertion site for foreign peptidesequences.

9. VP3 Capsid Assembly can be Achieved in Insect Cells 9.1. Cloning ofthe VP1 Mutant “Modification 4”

The construct pVL_VP1_MOD4 was generated to produce viral particlesconsisting essentially of the capsid protein VP3 in the absence of anyRep expression.

In detail, pUC19AV2 (described in detail in U.S. Pat. No. 6,846,665) wasused as template to amplify VP1 according to standard PCR conditions inthe presence of the following primers:

Insect_mod_4_s: (SEQ ID NO: 77) 5′-CAC CCG CGG GGA TCC GCC GCT GCC GACGGT TAT CTA CCC GAT TGG CTC-3′, and E_VP2_rev: (SEQ ID NO: 78) 5′-CGCGAA TTC CTA TTA CAG ATT ACG AGT CAG G-3′

Thereby, the wildtype translation start codon ATG (coding for Methionin)of VP1 was changed into GCC (Alanin) and inactivated. The resultingEcoRI/BamHI fragment was cloned into pBSIIKS (Stratagene, La Jolla,Calif., USA). This vector was used to inactivate the translation startcodon of VP2 by site directed mutagenesis according to the instructionsof the QuickChange II Site directed mutagenesis kit (Stratagene) usingthe following primers:

Insect-muta_4_s: (SEQ ID NO: 79) 5′-ACC TGT TAA GAC AGC TCC GGG AAA AAAG-3′ Insect-muta_4_as: (SEQ ID NO: 80) 5′-CTT TTT TCC CGG AGC TGT CTTAAC AGG T-3′

Thereby, the wildtype translation start codon ACG of VP2 was changedinto ACA (both coding for Threonin). The resulting construct wasdigested with restriction enzymes BamHI and EcoRI and cloned into thebaculo transfer vector pVL1393. As a result, the construct contained thecomplete AAV cap gene with mutations of the VP1 and VP2 start codons butno rep cds. (FIGS. 12A-12C)

9.2. Cloning of pVL_VP2

AAV2 VP2 was amplified using the primers E_VP2_for and E_VP2_rev listedbelow. Thereby, the wildtype VP2 translation start codon ACG (coding forThreonine) was changed into ATG (Methionine). Primers:

E_VP2_for: (SEQ ID NO: 81) 5′-CAC CCG CGG GGA TCC ACT ATGGCT CCG GGA AAAAAG AGG-3′ E_VP2_rev: (SEQ ID NO: 82) 5′-CGC GAA TTC CTA TTA CAG ATT ACGAGT CAG G-3′

The resulting construct was cloned into the baculo transfer vectorpVL1393.

9.3. Cloning of pVL_VP3

AAV2 VP3 was amplified using the primers E_VP3_for and E_VP3_rev listedbelow. Primers:

E_VP3_for: (SEQ ID NO: 83) 5′-CAC CCG CGG GGA TCC ACT ATG GCT ACA GGCAGT GGC GCA C-3′ E_VP2_rev: (SEQ ID NO: 84) 5′-CGC GAA TTC CTA TTA CAGATT ACG AGT CAG G-3′

The resulting construct was cloned into the baculo transfer vectorpVL1393.

9.4. Analysis of Particle Production

AAV particles were produced as described in 1.1. Cell lysates wereinvestigated by Western blot analysis for protein expression.pVL_VP1_MOD4 showed only VP3 expression, pVL_VP2 VP2 expression, whilepVL_VP3 showed in addition to VP3 smaller degradation signals (FIG.12B). Titers were obtained by an A20 ELISA. A titer of 1×10¹²particles/ml was observed for the modification 4 construct while VP2pVL_VP2 showed a titer of 9×10⁸ particles/ml and pVL_VP3 only a titer of1×10⁸ particles/ml (FIG. 12C).

Conclusion

This result shows that AAV VLPs can be produced in insect cells asefficiently as in mammalian cells. The data show that in insect cellsthe N-terminal sequence of VP3 also seems to be required and sufficientfor efficient VP3 capsid assembly. Further a change of the VP2 startcodon from ACG into ATG comes along with loss of efficiency in capsidassembly (FIG. 12C). We speculate that particle assembly from pVL_VP2goes along with minor VP3 expression initiated from a VP3 ATG which wasleft intact in the construct.

10. Capsids Composed Essentially of VP3 Tolerate Insertions ofPolypeptides 10.1. Generation of Virus-Like Particles (VLP) ContainingEpitopes at Position I-587

For cloning of expression vectors encoding VLPs composed of VP3 capsidproteins containing a particular epitope sequence at position I-587, theepitope sequence was first cloned into the VP coding sequence of pUCAV2at the site corresponding to I-587 (amino acid number relative to theVP1 protein of AAV-2) and was subsequently sub-cloned into the vectorpCIVP2mutACG.

Generation of vector pUCAV2 is described in detail in U.S. Pat. No.6,846,665. Basically, this vector contains the complete AAV2 genome(BgIII fragment) derived from pAV2 (Laughlin et al., 1983) cloned intothe BamHI restriction site of pUC19. pUCAV2 was further modified byintroduction of a NotI and AscI restriction site allowing the insertionof epitope sequences at position I-587 of the AAV2 capsid(PCT/EP2008/004366). In addition, an FseI restriction site locatedbetween position 453 (amino acid number relative to the VP1 protein ofAAV-2) and I-587 was introduced in-frame into the VP coding sequence ofthe vector by site directed mutagenesis.

For cloning of epitope sequences into modified pUCAV2 sense- andanti-sense oligonucleotides were designed that encode the respectiveepitope with an alanine or glycine adaptor sequence and contain a5′-site extension. The 5′-site extension of the oligonucleotides wasdesigned so that annealing of the sense and anti-sense oligonucleotidesresults in a dsDNA with 5′-site and 3′-site overhangs compatible withoverhangs generated by NotI and AscI restriction of the modified pUCAV2.The sequences of the oligonucleotides and the respective epitopesequences are summarized in Table 4. Each of the inserted epitopes isflanked by an adaptor according to the following scheme (X_(n)represents the epitope sequence):

-   -   Type I adaptor: (Ala)₂-(Gly)₃-X_(n)-(Gly)₄-Ala    -   Type II adaptor: (Ala)₂-(Gly)₄-X_(n)-(Gly)₄-Ala    -   Type III adaptor: (Ala)₃-(Gly)₅-X_(n)-(Gly)₅-(Ala)₂    -   Type IV adaptor: (Ala)₅-X_(n)-(Ala)₅

To anneal the oligonucleotides 50.0 μg of the sense oligonucleotide and50.0 μg of the anti-sense oligonucleotide were mixed in a total volumeof 200 μl 1×PCR-Buffer (Qiagen) and incubated for 3 min at 95° C. in athermomixer. After 3 min at 95° C. the thermomixer was switched off andthe tubes were left in the incubator for an additional 2 h to allowannealing of the oligonucleotides during the cooling down of theincubator. To clone the annealed oligonucleotides into pUCAV2 at I-587the vector was linearized by restriction with NotI and AscI and thecloning reaction was performed using the Rapid DNA Ligation Kit (Roche).Briefly, the annealed oligonucleotides were diluted 10-fold in 1×DNADilution Buffer and incubated for 5 min at 50° C. 100 ng of the annealedoligonucleotides and 50 ng of the Not//AscI linearized vector pUCAV2were used in the ligation reaction, which was performed according to theinstructions of the manufacturer of the Rapid DNA Ligation Kit (Roche).E. coli XL1 blue or DH5a were transformed with an aliquot of theligation reaction and plated on LB-Amp agar plates. Plasmids wereprepared according to standard procedures and were analyzed bysequencing.

For generation of empty VLPs composed of VP3 proteins containing anepitope sequence at I-587 the BsiWI/XcmI restriction fragment of pUCAV2containing the epitope at I-587 was sub-cloned into the vectorpCIVP2mutACG according to standard procedures. The vector pCIVP2mutACGcontains the overlapping AAV2 VP2 and VP3 coding sequences cloned intothe XhoI/NotI site of pCI (Promega). In pCIVP2mutACG the ACG start-codonof VP2 is destroyed and replaced by a GAG codon. Substitution wasperformed by PCR amplification of the AAV2 VP2 and VP3 coding sequencesusing VP2 specific primers and the plasmid pCIVP2 as template (thevector pCIVP2 contains the wildtype VP2 and VP3 coding sequence clonedinto the polylinker of pCI). The forward primer used for PCR anneals tothe 5′ site of the VP2 coding sequence and contains the substitution ofthe VP2 ACG start codon by a GAG codon. In addition, the forward primercontains an XhoI recognition sequence at the 5′-site. The reverse primerannealed to the 3′ end of the VP2/VP3 coding sequence and contained aNotI recognition sequence at its 5′-site. The resulting PCR product wascloned into the XhoI/NotI site of pCI.

The resulting vectors were used for production of VLPs by transfectionof 293-T cells. Cells (5×10⁵/dish) were seeded in 6 cm dishes 24 h priorto transfection. 293-T cells were transfected by calcium phosphateprecipitation as described in US 2004/0053410. Subsequently, 293-T cellswere lysed in the medium by three rounds of freeze (−80° C.) and thaw(37° C.) cycles. The lysate (3 ml total volume) was cleared bycentrifugation and the VLP capsid titer was determined using acommercially available ELISA (AAV Titration ELISA; Progen, Heidelberg,Germany). VLP titers ranged between 2.1 E+12 and 9.8 E+12 capsids/ml(Table 5). The VLP TP18 clone was directly used for large scalepackaging (as described in example 1). It contained 1.2E+13 capsids/mlwithin the crude lysate (Table 5).

10.2. Generation of Virus-Like Particles (VLP) Containing Epitopes atPosition I-587 and I-453 of the Capsid

For cloning of expression vectors encoding VLPs composed of VP3 capsidproteins containing epitope sequences at position I-453 and I-587 (aminoacid number relative to the VP1 protein of AAV-2), the first epitopesequence was cloned into pCIVP2mutACG at the site corresponding to I-587as described above.

The second epitope sequence was initially cloned into the NotI/AscIrestriction site of the vector pCIVP2-I453-NotI-AscI (described in: WO2008/145400). Briefly, the vector pCI-VP2-I453-Not-AscI was created byPCR amplification of the AAV2 VP2 gene and cloning of the respective PCRproduct into the XhoI/NotI site of vector pCI (Promega). The resultingvector pCIVP2 was modified by destruction of the NotI restriction siteof the cloning site by site-directed mutagenesis. The vector was furthermodified by introduction of a novel singular NotI and AscI restrictionsite allowing the insertion of epitope sequences at position I-453 ofthe AAV2 capsid. In addition, an FseI site located between I-453 andI-587 was introduced in-frame into the VP coding sequence ofpCIVP2-I453-NotI-AscI by site directed mutagenesis. For cloning ofepitope sequences into the NotI/AscI site of the vector sense- andanti-sense oligonucleotides were designed that encode the respectiveepitope with a alanine/glycine adaptor sequence and contain a 5′-siteextension. The 5′-site extension of the oligonucleotides was designed sothat annealing of the sense and anti-sense oligonucleotides results in adsDNA with 5′-site and 3′-site overhangs compatible with overhangsgenerated by NotI and AscI restriction of pCIVP2-I453-Not-AscI. Cloningof the annealed oligonucleotides was performed as described above.

The sequences of the oligonucleotides and the respective epitopesequences are summarized in Table 6. Each of the inserted epitopes isflanked by an adaptor according to the following scheme (X_(n)represents the epitope sequence):

-   -   (Ala)₂-(Gly)₃-X_(n)-(Gly)₄-Ala

For generation of bivalent VLPs displaying epitopes (murine TNFα orIL-17 epitope) at I-453 and I-587 the BsiWI/FseI fragment ofpCIVP2-I453-NotI-AscI containing a given epitope inserted at I-453 wassubcloned into the vector pCIVP2mutACG containing a particular epitopeinserted into I-587 (described above). The resulting vector was used forproduction of bivalent VLPs by transfection of 293-T cells as describedabove (example 1.2) (6-well plate scale). Particle production wasanalyzed by ELISA (AAV2 Titration ELISA; Progen). Results are shown inTable 7. These data demonstrate that VLPs composed of VP3 proteins withepitope insertions at I-453 and I-587 can be produced with high capsidtiters.

TABLE 4 Oligonucleotides used for cloning of epitope sequences intoI-587 Name/ sense anti-sense Peptide Seq. Type OligonucleotideOligonucleotide Adaptor CETP TP18 Rabbit 5′GGCCGGCGGAGGTGACAT5′CGCGCACCGCCACCCCC Type I DISVTGAPVIT CETP CAGCGTGACCGGTGCACCCGCAGGTAGGTGGCGGTGATC ATYL epitope TGATCACCGCCACCTACCTGACGGGTGCACCGGTCACGC GGGGGTGGCGGTG 3′ TGATGTCACCTCCGCC 3′ (SEQ ID NO: 85)(SEQ ID NO: 86) 3Depi-3 Human 5′GGCCGGCGGAGGTGGTGA 5′CGCGCACCGCCACCCCCType II DSNPRGVSAY IgE CAGCAACCCTAGAGGCGTGA TCTGCTCAGGTAGGCGCTC LSRepitope GCGCCTACCTGAGCAGAGGG ACGCCTCTAGGGTTGCTGT GGTGGCGGTG 3′CACCACCTCCGCC 3′ (SEQ ID NO: 87) (SEQ ID NO: 88) Kricek Human5′GGCCGCAGCGGCGGTGAA 5′CGCGCCGCCGCCGCCGC Type IV VNLTWSRASG IgECCTGACCTGGAGCAGAGCCT GCCGGAGGCTCTGCTCCAG epitope CCGGCGCGGCGGCGGCGGGTCAGGTTCACCGCCGCTG 3′ (SEQ ID NO: 89) C3′ (SEQ ID NO: 90) TNFα-V1Murine 5′GGCCGGCGGAGGTAGCAG 5′CGCGCACCGCCACCCCC Type I SSQNSSDKPV TNFαCCAGAACAGCAGCGACAAGC CTCCACCTGGTGGTTAGCC AHVVANHQVE epitopeCCGTGGCCCACGTGGTGGCT ACCACGTGGGCCACGGGCT AACCACCAGGTGGAGGGGGGTGTCGCTGCTGTTCTGGCT TGGCGGTG 3′ GCTACCTCCGCC 3′ (SEQ ID NO: 91) (SEQ IDNO: 92) IL-17-V1 Murine 5′GGCCGGCGGAGGTAACGC 5′CGCGCACCGCCACCCCC Type INAEGKLDHH IL-17 CGAGGGCAAGCTTGACCACC CAGCACGCTGTTCATGTGG MNSVL epitopeACATGAACAGCGTGCTGGGG TGGTCAAGCTTGCCCTCGG GGTGGCGGTG 3′ CGTTACCTCCGCC 3′(SEQ ID NO: 93) (SEQ ID NO: 94) IL-6-V2 Murine 5′GGCCGGCGGAGGTCTGGA5′CGCGCACCGCCACCCCC Type I LEEFLKVTLRS IL-6 GGAATTCCTGAAGGTGACCCGCTTCTCAGGGTCACCTTC epitope TGAGAAGCGGGGGTGGCGGT AGGAATTCCTCCAGACCTC G3′ CGCC 3′ (SEQ ID NO: 95) (SEQ ID NO: 96) Aβ(1-9) Human5′GGCCGCAGGCGGAGGGGG 5′CGCGCCGCGCCTCCCCC Type III DAEFRHDSG amyloid-AGGCGACGCCGAGTTCAGAC TCCGCCGCCGCTGTCGTGT β epitope ACGACAGCGGCGGCGGAGGGCTGAACTCGGCGTCGCCTC GGAGGCGCGG 3′ CCCCTCCGCCTGC 3′ (SEQ ID NO: 97) (SEQID NO: 98)

TABLE 5 Small scale production of different VLPs Titer (capsids/ NameEpitope at I-587 ml) VLP-TP18 CETP TP18 1.2E+13(*) DISVTGAPVITATYL (SEQID NO: 99) VLP- 3Depi-3 2.1E+12 3Depi3 DSNPRGVSAYLSR (SEQ ID NO: 100)VLP- Kricek 2.6E+12 Kricek VNLTWSRASG (SEQ ID NO: 101) VLP-TNFα TNFα-V19.8E+12 SSQNSSDKPVAHVVANHQVE (SEQ ID NO: 102) VLP-IL- IL-17-V1 5.6E+1217 NAEGKLDHHMNSVL (SEQ ID NO: 103) VLP-IL-6 IL-6-V2 5.6E+12 LEEFLKVTLRS(SEQ ID NO: 104) VLP-Aβ Aβ(1-9) 6.2E+12 DAEFRHDSG (SEQ ID NO: 105)(*)Large-scale packaging

TABLE 6 Oligonucleotides used for cloning of epitope sequences intoI-453 Name/ sense anti-sense Peptide Seq. Type OligonucleotideOligonucleotide TNFα-V1 Murine 5′GGCCGCCGGTGGAGGCAG5′CGCGCCCTCCACCGCCCTCCAC SSQNSSDKPVA TNFα CAGCCAGAACAGCAGCGACACTGGTGGTTAGCCACCACGTGGGC HVVANHQVE epitope AGCCCGTGGCCCACGTGGTGCACGGGCTTGTCGCTGCTGTTCTG GCTAACCACCAGGTGGAGGG GCTGCTGCCTCCACCGGC 3′CGGTGGAGGG 3′ (SEQ ID NO: 107) (SEQ ID NO: 106) IL-17-V1 Murine5′GGCCGCCGGTGGAGGCAA 5′CGCGCCCTCCACCGCCCAGCAC NAEGKLDHHMN IL-17CGCCGAGGGCAAGCTTGACC GCTGTTCATGTGGTGGTCAAGCTT SVL epitopeACCACATGAACAGCGTGCTG GCCCTCGGCGTTGCCTCCACCGGC GGCGGTGGAGGG 3′ 3′ (SEQ IDNO: 108) (SEQ ID NO: 109) IL-6-V2 Murine 5′GGCCGCCGGTGGAGGCCT5′CGCGCCCTCCACCGCCGCTTCT LEEFLKVTLRS IL-6 GGAGGAATTCCTGAAGGTGACAGGGTCACCTTCAGGAATTCCTC epitope CCCTGAGAAGCGGCGGTGGA CAGGCCTCCACCGGC 3′GGG 3′ (SEQ ID NO: 110) (SEQ ID NO: 111)

TABLE 7 Production of VLPs carrying epitopes at I-453 and I-587 Titer(capsids/ combination Epitope at I-453 Epitope at I-587 ml) TNF-α/IL-17TNF α-V1 IL-17-V1 7.9E+12 SSQNSSDKPVAHVVANHQVE NAEGKLDHHMNSVL (SEQ IDNO: 112) (SEQ ID NO: 113) TNF-α/IL-6 TNF α-V1 IL-6-V2 8.5E+12SSQNSSDKPVAHVVANHQVE LEEFLKVTLRS (SEQ ID NO: 114) (SEQ ID NO: 115)IL-17/TNF-α IL-17-V1 TNFα-V1 1.0E+13 NAEGKLDHHMNSVL SSQNSSDKPVAHVVANHQVE(SEQ ID NO: 116) (SEQ ID NO: 117) IL-6/TNF-α IL-6-V2 TNFα-V1 1.0E+13LEEFLKVTLRS SSQNSSDKPVAHVVANHQVE (SEQ ID NO: 118) (SEQ ID NO: 119)IL-17/IL-6 IL-17-V1 IL-6-V2 3.9E+12 NAEGKLDHHMNSVL LEEFLKVTLRS (SEQ IDNO: 120) (SEQ ID NO: 121) IL-6/IL-17 IL-6-V2 IL-17-V1 8.9E+12LEEFLKVTLRS NAEGKLDHHMNSVL (SEQ ID NO: 122) (SEQ ID NO: 123)

10.3. Conclusion

VP3 particles tolerate insertions and can therefore be used as amedicament such as a vaccine for example by insertion of B-Cellepitopes.

11. VP3 Capsid Assembly of Different AAV Serotypes 11.1. AAV1 DeletionConstructs

To analyze whether these findings can be conferred to other serotypes ananalogue setting of constructs for AAV1 were tested.

Following constructs were cloned:

-   -   pCI_VP2/2539_AAV1: The complete AAV1 VP2 plus 95 bp of VP1 were        cloned into pCI (Promega, Mannheim, Germany). The VP2 ACG start        codon was not mutated.    -   pCI_VP3/2539_AAV1 mutACG: The complete AAV1 VP2 plus 95 bp of        VP1 were cloned into pCI. The VP2 ACG start codon was mutated to        ACC.    -   pCI_VP3/2634_AAV1 mutACG: The VP1 part was deleted completely        and the VP2 ACG start codon was mutated into an ACC.

Cloning

Cloning of all constructs was performed by site directed mutagenesisstandard procedures using modified primers (primers used for sitedirected mutagenesis are listed below). pCI_VP2/2539_AAV1 was generatedby inserting a NheI site 95 bp upstream of the VP2 ACG start codon and aXmaI site downstream of the VP3 stop codon. Mutations were generatedwithin pUCrep/fs/cap_AAV1_1588 (described within PCT/EP2008/004366). Theresulting plasmid was digested with NheI and XmaI. The generatedfragment was cloned into the pCI-VP2 Vector (described inPCT/EP2008/004366). Primers:

AAV1 NheI VP2plus95bp: (SEQ ID NO: 124) 5′-GAG CGT CTG CTA GCA GAT ACCTCT TTT GGG G-3′ AAV1 VP3 Xma rev: (SEQ ID NO: 125) 5′-GAA ACG AAT CACCCG GGT TAT TGA TTA AC-3′pCI_VP3/2539_AAV1 mutACG was generated by mutating the ACG start codonto ACC within pCIVP2/2539_AAV1. Primer:

AAV1 VP2ko for: (SEQ ID NO: 126) 5′-GGC GCT AAG ACC GCT CCT GGA AAG-3′AAV1 VP2ko rev: (SEQ ID NO: 127) 5′-CTT TCC AGG AGC GGT CTT AGC GCC-3′pCI_VP3/2634_AAV1 mutACG was generated by deleting the 95 bp directlyupstream of the VP2 ACG start codon and mutating by the same step theACG start codon to ACC within pCIVP2_AAV1. Primer:

AAV1 VP2ko_VP1del for: (SEQ ID NO: 128) 5′-ACG ACT CAC TAT AGG CTA GCAGGC GCT AAG ACC GCT CCT GGA AAG-3′ AAV1 VP2ko_VP1del rev: (SEQ ID NO:129) 5′-CTT TCC AGG AGC GGT CTT AGC GCC TGC TAG CCT ATA GTG AGT CGT-3′

Assembly of AAV1 capsids was controlled within crude lysates aftertransfection of 293 cells with the respective plasmid. The capsid titerwas determined by an AAV1 titration ELISA (Progen, Heidelberg, Germany)according to manufacturer's manual. The assembly efficiency of the threeAAV1 constructs was comparable. The construct pCI_VP3/2634_AAV1 mutACGgave a titer of 10¹³ particles/ml, confirming the fact that capsidgeneration of AAV1 particles is generally more efficient than of AAV2particles. In Western blot analyses VP2 and VP3 proteins were detectablefor construct pCI_VP2/2539_AAV1 and only VP3 was detectable forpCI_VP3/2539_AAV1 mutACG and pCI_VP3/2634_AAV1 mutACG respectively (FIG.13).

As a control for capsid protein expression, pUCAV1 was transfected.pUCAV1 contains the complete AAV1 Cap open reading frame encoding VP1,VP2 and VP3 of AAV1. pUCAV1 is described in detail in the PCT submissionPCT/EP2008/004366 (there referred to as “pUCAV1_AgeI”).

11.2. Trans-Complementation of pCMV Driven AAV1 VP3 Constructs

To see whether trans-complementation experiments described in example 5can be conferred to other serotypes analogue constructs of pCMV-VP3/2809(AAV2) were cloned for AAV1.

11.2.1. Cloning

pCMV_AAV1VP3/2829 was cloned as following: By mutagenesis a HindIIIrestriction site was introduced directly before the VP3 ATG start codonof plasmid pUCrep/fs/cap_AAV1 (described within PCT/EP2008/004366) usingthe primers indicated below. The resulting plasmid was digested withAgeI. The Age I site was blunt ended with Klenow polymerase and theconstruct was subsequently digested with HindIII. The generated fragmentwas cloned into the HindIII/HincII-digested pBSCMV backbone. pBSCMV wasgenerated by insertion of a 650 bp BamHI CMV promoter fragment into theBamHI site of BlueskriptII SK+ vector (Stratagene, Amsterdam,Netherlands) described by Wistuba et al, 1997. Primer Hind IIImutagenesis:

Forward: (SEQ ID NO: 130) 5′-CGC TGC TGT GGG ACC TAA GCT TAT GGC TTC AGGCGG TGG CG-3′ Reverse: (SEQ ID NO: 131) 5′-CGC CAC CGC CTG AAG CCA TAAGCT TAG GTC CCA CAG CAG CG-3′

11.2.2. Trans-Complementation Assay

Trans-complementation was performed with the pVP2N-gfp construct fromAAV2 as described in example 3. Cells were transfected with plasmidpCMV-VP3 of either AAV2 pCMV_VP3/2809) or AAV1 (pCMV_AAV1VP3/2829) withor without cotransfection of pVP2N-gfp (FIG. 14). Same molar ratios ofVP3 construct and pVP2N-gfp were transfected. Protein expression wasanalyzed by Western blot and particle formation efficiency was measuredby ELISA.

11.2.3. Result and Conclusion

Particle assembly of AAV1 analyzed by an AAV1 ELISA (Progen, Heidelberg)was rescued by trans-complementation with pVP2N-gfp derived from AAV2.Rescue efficiency cannot be indicated as we did not comparecotransfection of pCMV_AAV1VP3/2829 and pVP2N-gfp with transfection ofpCIVP3/2634_AAV1 mutACG (see chapter 11.1 above). Also, we did not yetclone and test an AAV1 trans-complementation plasmid pVP2N-Gfp

Particle titer measured for trans-complemented AAV2 VP3 was 2.1E11. ForAAV1 VP3 the titer obtained was 3.4E10 (a direct comparison of AAV1 andAAV2 titers is not possible due to the use of different ELISAs).

The results indicate that AAV1 makes use of the same mechanism forcapsid assembly as AAV2 and that fragment Z and VP3 are interchangeablewith different AAV serotypes.

11.3. Insertion of Polypeptides within AAV1 I588 is Tolerated

Here it was investigated whether empty AAV1 essentially VP3 particlestolerate insertions within amino-acid position 588.

For cloning of epitope sequences into pUCAV1-AgeI-1588 (described inPCT/EP2008/004366), sense- and anti-sense oligonucleotides were designedthat encode the respective epitope with a glycine adaptor sequence. Uponhybridization of both oligonucleotides, 5′- and 3′-overhangs aregenerated that are compatible with overhangs generated by NotI and AscIrestriction of the pUCAV1-AgeI-1588. The sequences of theoligonucleotides and the respective epitope sequences investigated aresummarized in Table 4. Each of the inserted epitopes is flanked by anadaptor according to the following scheme (X_(n) represents the epitopesequence): Ser(588)-(Ala)₂-(Gly)₅-X_(n)-(Gly)₅-Thr(589)

Oligo nucleotides for cloning the human IgE epitope “Kricek”

Amino acid sequence: VNLTWSRASG Sense oligo: (SEQ ID NO: 132) 5′-g gccgca gcc gca gtg aac ctg acc tgg agc aga gcc tcc ggc gcg gca gct gcagct-3′ antisense oligo: (SEQ ID NO: 133) 5′-g gcg agc tgc agc tgc cgcgcc gga ggc tct gct cca ggt cag gtt cac tgc ggc tgc-3′

Oligo nucleotides for cloning the human IgE epitope “3Depi-3”

Amino acid sequence: DSNPRGVSAYLSR Sense oligo: (SEQ ID NO: 134) 5′-GGCCGGC GGT GGA GGC GGT GAC AGC AAC CCT AGA GGC GTG AGC GCC TAC CTG AGC AGAGGA GGC GGT GGA GGG-3′ antisense oligo: (SEQ ID NO: 135) 5′-CGCG CCC TCCACC GCC TCC TCT GCT CAG GTA GGC GCT CAC GCC TCT AGG GTT GCT GTC ACC GCCTCC ACC GCC-3′

The precise cloning procedure used corresponds to the protocol used forinsertion of epitopes into AAV2 I587 described in example 10.

For generation of empty AAV1 VLPs composed of essentially VP3 proteinscontaining an epitope sequence at I-588 the BsiWI/SphI restrictionfragment of pUCAV1-AgeI-1588 carrying the epitope at I-588 wassub-cloned into the vector pCIVP3/2634_AAV1 mutACG (described in example11.1) according to standard procedures.

The resulting vectors were used for production of AAV1 VLPs bytransfection of 293-T cells as described above (example 1.2.)

Titers were determined by a commercial AAV1 ELISA (Progen, Heidelberg,Germany). High titers of 3.6E13/ml (Kricek) and 9.2E13/ml (3Depi-3) wereobtained, indicating that insertions within AAV1 588 (being homologousto AAV2 587) are well tolerated and that AAV1 VP3 particles can be usedas vaccine carrier.

12. ORF2 Comprises Fragment Z and Encodes AAP.

Detailed sequence analysis revealed that fragment Z encodes asignificant part of the new “assembly activating protein” (AAP). FIG. 16gives an overview and FIG. 17 shows in more detail the position of ORF2and the encoded protein AAP in relation to the cap gene and the positionof the translation start codons of the Cap proteins VP1, VP2 and VP3, aswell as the location of fragment Z and EcoNI and BsiWI restrictionsites. The three Cap proteins VP1, VP2 and VP3 are translated from thesame one ORF of the cap gene (also named the first ORF, ORF1), whereasAAP is translated from a different reading frame (named the second ORF,ORF2). For VP1, VP2 and VP3 numbers of the well-defined translationstart points are given, whereas for AAP it is not definitely known.

In FIG. 17 the sequence of ORF2 (627 nucleotides, SEQ ID NO: 23) and therespective AAP protein sequence (208 amino acids, SEQ ID NO: 1) is givenfor AAV2 as extracted from NCBI entrée number NC 001401.

The sequences of the respective open reading frames and proteins of someother parvoviruses were extracted from the capsid gene sequencesavailable in the NCBI database and given in detail in SEQ ID Nos 2-44 aslisted in table 8.

TABLE 8 NCBI entrée numbers and numbers of corresponding SEQ IDs of AAPencoding nucleotide and protein sequences from different parvoviruses.No. of nt respective Length of encoded protein Length of parvovirusentrée at NCBI ORF2 ORF2/nt AAP AAP/AA AAV2 NC_001401 SEQ ID NO: 23 627SEQ ID NO: 1 208 AAV1 NC_002077 SEQ ID NO: 24 678 SEQ ID NO: 2 225 AAV3bAF028705 SEQ ID NO: 25 627 SEQ ID NO: 3 208 AAV4 NC_001829 SEQ ID NO: 26597 SEQ ID NO: 4 198 AAV5 NC_006152 SEQ ID NO: 27 681 SEQ ID NO: 5 226AAV6 AF028704 SEQ ID NO: 28 678 SEQ ID NO: 6 225 AAV7 NC_006260 SEQ IDNO: 29 681 SEQ ID NO: 7 226 AAV8 NC_006261 SEQ ID NO: 30 684 SEQ ID NO:8 227 AAV9 AY530579 SEQ ID NO: 31 681 SEQ ID NO: 9 226 AAV10 AY631965SEQ ID NO: 32 606 SEQ ID NO: 10 201 AAV11 AY631966 SEQ ID NO: 33 594 SEQID NO: 11 197 AAV12 DQ813647 SEQ ID NO: 34 621 SEQ ID NO: 12 206 b-AAV(bovine) NC_005889 SEQ ID NO: 35 600 SEQ ID NO: 13 199 Avian AAVAY186198 SEQ ID NO: 36 789 SEQ ID NO: 14 262 ATCC VR-865 Avian AAVAY629583 SEQ ID NO: 142 723 SEQ ID NO: 143 240 strain DA-1 AAV13EU285562 SEQ ID NO: 37 627 SEQ ID NO: 15 208 Mouse AAV1 DQ100362 SEQ IDNO: 38 534 SEQ ID NO: 16 177 Avian AAV AY629583 SEQ ID NO: 39 723 SEQ IDNO: 17 240 strain DA-1 Caprine AAV1 AY724675 SEQ ID NO: 40 581 SEQ IDNO: 18 226 isolate AAV-Go. 1 Rat AAV1 DQ100363 SEQ ID NO: 41 756 SEQ IDNO: 19 251 Goose EU088102 SEQ ID NO: 42 639 SEQ ID NO: 20 212 parvovirusstrain DB3 Duck AY382892 SEQ ID NO: 43 693 SEQ ID NO: 21 230 parvovirusstrain 90-0219 Snake AY349010 SEQ ID NO: 44 600 SEQ ID NO: 22 199parvovirus 1

For sequence comparison an alignment of the predicted AAP proteinsequences derived from ORF2 of the cap gene of some parvoviruses isgiven in FIGS. 27A and 27B.

In construct pVP2N-gfp the EcoNI/BsiWI fragment from pTAV2.0 wasinserted downstream of a CMV promoter and upstream of the GFP cds ofvector pEGFP-N1 (example 3.1/FIG. 6A and example 13/FIG. 19A). Since theBsiWI site is located about 90 nucleotides upstream of the 3′ end ofORF2, the vector pVP2N-gfp encodes C-terminally truncated AAP (namedAAPtru) that is as active in trans-complementation as AAP expressed fromfull-length ORF2 (see e.g. FIGS. 21A-21C).

13. Codon Modification Confirms that Expression of Functional Proteinfrom ORF2 is Necessary for Trans-Complementation

To investigate the nature of the trans-complementing activity of ORF2,the sequence between the EcoNI/BsiWI restriction fragment was codonmodified (cm).

The first mutant DNA sequence was named ORF1 cm. The DNA sequence of themutant was altered in such a way that the first reading frame coding forthe capsid protein remained intact whereas the second reading framecoding for AAP was changed. As a result the sequence encodes wildtypecapsid protein but no functionally active AAP any more. Identity of theDNA sequence of pVP2N-gfp versus pVP2N/ORF1cm-gfp is 71% while proteinidentity in the first reading frame is 100%.

The second mutant DNA sequence was named ORF2 cm and altered in thefirst reading frame meaning that it did not code for a functionallyactive capsid protein any more but functionally intact AAP could beexpressed. Identity of the DNA sequence of pVP2N-gfp versus pVP2N/ORF2cm-gfp is 79% while protein identity in the second reading frame is100%.

The sequences of ORF1cm and ORF2 cm are given in FIGS. 18A and 18B,respectively. As already described in example 5, codon modification wasperformed by GENEART (Regensburg, Germany). Codons were modified forcodons preferentially used in mammalian cells.

As described in example 3.1, pVP2N-gfp was generated by inserting theEcoNI/BsiWI restriction fragment of pTAV2.0 into the multiple cloningsite of pEGFP-N1. Constructs pVP2N/ORF1cm-gfp and pVP2N/ORF2 cm-gfp weregenerated in the same way with the difference that the codon modifiedEcoNI/BsiWI fragments were inserted into the corresponding vectorbackbone.

Protein expression of pVP2N/ORF1cm-gfp and pVP2N/ORF2 cm-gfp (FIG. 20A)was compared with that of unmodified pVP2N-gfp (FIG. 20B) in Westernblot analysis. The ability to rescue capsid formation of pCMV-VP3/2809was tested in trans-complementation assays as described in example 3.Plasmids were cotransfected in a molar ratio of 1:1 (FIG. 20C).

Result and Conclusion

As already described in example 3 and shown in FIGS. 6A-6D, Western blotanalysis using monoclonal antibody A69 confirmed expression of a capsidprotein comprising the VP2 N-terminus (VP2N-gfp, FIG. 19B) in the GFPfusion construct pVP2N-gfp (FIG. 19A). Complementation of plasmidpCMV-VP3/2809 with different molar ratios of pVP2N-gfp in 293-T cellscorresponding to decreasing amounts of co-transfected pVP2N-gfp showeddecreasing capsid assembly upon its quantification (FIG. 19C).Determination of the number of assembled capsids also revealed thatdeletion mutant pCMV-VP3/2809 co-transfected with pVP2N-gfp was nearlyas efficient in capsid assembly as mutant pCMV-VP3/2696, the deletionmutant that showed normal capsid formation (FIGS. 5A-5D). Assembly couldbe detected even at a 500-fold reduced amount of co-transfectedpVP2N-gfp plasmid.

Hence it was clear, that the assembly promoting activity associated withthe constructs containing cap sequences upstream of the VP3 translationstart site can be provided in trans.

As already described for example 5, FIG. 8D codon-modified constructpVP2N/ORF1cm protein expression from codon-modified constructs was evenhigher than protein expression from the non-modified constructpVP2N-gfp, since the codon modification was optimized for mammaliancells. VP3 levels from co-expressed pCMV-VP3/2809 were normal. However,capsid assembly was not detected when using the helper constructpVP2N/ORF1 cm (FIG. 20C). Also reduced expression of the respectiveprotein by transfecting lower amounts of pVP2N/ORF1 cm did not supportcapsid formation of VP3 (data not shown).

In contrast, assembled capsid could be detected using the helperconstruct pVP2N/ORF2 cm (FIG. 20C). As described above, only ORF2 cmexpresses functionally intact AAP, whereas in pVP2N/ORF1 cm the sequenceof AAP is non-functional and this codon-modified construct encodessolely capsid protein. Accordingly, only pVP2N/ORF2 cm rescued capsidassembly in trans-complementation.

This result clearly indicates, that the trans-complementing activity offragment Z is mediated by its encoded protein AAP in ORF2. Codonmodification experiments confirmed that expression of functional capsidprotein in ORF1 is not necessary for trans-complementation butexpression of functional AAP in ORF2.

14. Mutation of the Predicted Translation Start Codon of AAP

The sequence of ORF2 as given in FIG. 17 was analyzed in detail tofurther characterize AAP mediating capsid assembly. ORF2 does notcontain an ATG prior to the VP3 start codon. It has to be assumed that anon-canonical start codon is utilized which is upstream of the definedminimal 5′-end of fragment Z at nt 2765. Taken into account the sequencerequirements in the local environment of a start codon i.a. as definedby Kozak (2002) we predict the fifth codon at position 2729-2731, whichis CTG and encodes a leucine (underlined in FIG. 17), to be thenon-canonical start codon for translation of AAP. To observe itsinfluence on expression efficiency, the site was mutated into ATG andTTG.

Protein expression of AU1 tagged versions of ORF2, namely pORF2/CTG-AU1,pORF2/ATG-AU1 and pORF2/TTG-AU1 (FIG. 21A), was compared with that ofunmodified pVP2N-gfp in Western blot analysis (FIG. 21B). The ability torescue capsid formation of pCMV-VP3/2809 was tested intrans-complementation assays as described in example 3. Plasmids werecotransfected in a molar ratio of 1:1 (FIG. 21C).

Constructs pORF2/CTG-AU1, pORF2/ATG-AU1 and pORF2/TTG-AU1 comprise theentire ORF2 of the cap gene (AAV2 nt 2717-3340) fused to sequencescoding for an AU1-tag (FIG. A).

For generation of constructs pORF2/CTG-AU1, pORF2/ATG-AU1 andpORF2/TTG-AU1 PCRs were performed with template pTAV2.0 and forwardprimer

(SEQ ID NO: 136) 5′-GGATCGCAAGCTTATTTTGGTCAGACTGGAGACGCAGACTCAGTACCTGACCC-3′, (SEQ ID NO: 137)5′-GGATCGCAAGCTTATTTTGGTCAGAATGGAGACGCAGACTCAG-3′,or

(SEQ ID NO: 138) 5′-GGATCGCAAGCTTATTTTGGTCAGATTGGAGACGCAGACTCAG-3′and reverse primer

(SEQ ID NO: 139) 5′-GCGGTGTCTCGAGTTATATATAGCGATAGGTGTCGGGTGAGGTATCCATACTGTGGCACCATGAAGAC-3′.

The HindIII/XhoI digested amplification products were inserted into theHindIII/XhoI backbone of pBS-CMVsense, which was generated by insertionof a 560 bp BamHI human cytomegalovirus (CMV) promoter fragment frompHCMV-Luci (kindly provided by K. Butz, Germen Cancer Research Center,Heidelberg, Germany) into the BamHI site of plasmid Bluescript IISK+(pBS, Stratagene, La Jolla, Calif., USA).

Results and Conclusion

The expression of the postulated proteins could be demonstrated using amonoclonal antibody against the AU1-tag (anti-AU1) for the constructspORF2/CTG-AU1 and pORF2/ATG-AU1 (FIG. 21B), whereas expression fromconstruct pORF2/TTG was below the detection level. Co-transfection ofthe ORF2 containing plasmids pORF2/CTG-AU1, pORF2/ATG-AU1 andpORF2/TTG-AU1 with the VP3 expression plasmid pCMV-VP3/2809 yieldedcapsid formation (FIG. 21C) wherein the number of assembled capsidsmeasured per volume correlated with the amount of expressed proteinestimated from the Western blot. Capsid titers obtained aftertransfection of pORF2/ATG-AU1 with pCMV-VP3/2809 were comparable tothose obtained after co-transfection of pVP2N-gfp with pCMV-VP3/2809. Incontrast, the TTG start codon encoding plasmid stimulated capsidassembly by a factor of approximately 10³ fold less compared to thepVP2N-gfp plasmid. A polyclonal antiserum directed against a peptide ofORF2 clearly indicated expression of AAP and detected in addition to theAU1-tagged full length AAP also the C-terminally truncated AAP (AAPtru)expressed from pVP2N-gfp (FIG. B).

Taken together, mutation of the putative non-canonical CTG start codoninto a strong ATG start codon enhanced protein synthesis and capsidassembly whereas mutation into a codon which normally is not preferredas initiation codon for protein synthesis significantly reduces proteinlevels and the number of assembled capsids. This result not onlycorroborates our conclusion that the protein product of ORF2 promotesthe capsid assembly process. The results further indicate that thenon-canonical CTG start codon is likely used as a start for translation,as its mutation into TTG leads to a significant reduction of AAPexpression.

15. Insertion of Stop Codons in ORF2 Confirm that Expression ofFunctional AAP is Necessary for Trans-Complementation

Additionally, mutations were performed in the AAP encoding reading frameby introduction of stop codons into ORF2 in order to confirm thatexpression of functional AAP is necessary for trans-complementation.

Plasmids pVP2N/ORF2stopA-gfp, pVP2N/ORF2stopB-gfp, andpVP2N/ORF2stopC-gfp were created by site-directed mutagenesis(QuickChange site-directed mutagenesis kit, Stratagene) of templatepVP2N-gfp using two complementary PCR primers which included the desiredsubstitutions. In pVP2N/ORF2stopA-gfp codon tgg₂₈₁₁ has been mutatedinto tag, in pVP2N/ORF2stopB-gfp codon c₂₈₃₁aa has been mutated intotaa, and in pVP2N/ORF2stopC-gfp codon g₂₈₇₉aa has been mutated into tga(FIG. 22A). Positions are according to Ruffing et al. (1994). Allmutations do not disrupt ORF1. In each case the EcoNI/BsiWI fragment wasthen cloned into the EcoNI/BsiWI backbone of pVP2N-gfp.

Results and Conclusion

Western blot analysis confirmed that VP3 is expressed in all samples(detected by monoclonal antibody B1 in FIG. 22B). Again, Bluescriptvector (pBS) did not cause capsid assembly in the trans-complementationassay (FIG. 22C). Introduction of stop codons into ORF2 of the cap geneat the three different sites (as indicated in FIG. 22A) did notinfluence expression of VP2N-gfp (FIG. 22B), whereas all mutantsharboring stop codons in ORF2 did not show any activity in capsidassembly (FIG. 22C).

Accordingly, Cap expression from pVP2n-gfp is not sufficient for capsidassembly in the trans-complementation assay. This result clearlysupports the existence of AAP expressed from a different reading frame(ORF2) overlapping with the cap gene, which provides the capsid assemblyhelper function.

16. Expression of Functional AAP Rescues Capsid Assembly in the Contextof the AAV Genome

Next we wanted to analyze whether expression of the newly discovered“assembly activating protein” AAP is necessary for capsid assembly inthe context of the whole AAV genome. Therefore, construct pTAV/ORF1cmwas created by cloning the EcoNI/BsiWI fragment of pVP2N/ORF1cm-gfp(example 13) into the EcoNI/BsiWI backbone of pTAV2.0 (example 1.2.1.).Hence, plasmid pTAV/ORF1cm (schematically shown in FIG. 23A) encodes theknown AAV2 capsid and Rep proteins but should be deficient in thesynthesis of AAP, because the codons of the cap gene were modified inthe second reading frame (ORF2) without changing the first one encodingthe Cap proteins (ORF1).

Results and Conclusion

Indeed, the four Rep proteins (Rep40, Rep52, Rep68, and Rep78) werecorrectly expressed (data not shown). Western blot analysis showed thatthe expression pattern of the three VP proteins was slightly altered.Expression of endogenous AAP from wildtype plasmid pTAV2.0 but not fromthe codon modified one pTAV/ORF1cm was directly proven using polyclonalanti-AAP serum (FIG. 23B). As expected, truncated AAP is detectable uponco-expression of pVP2N-gfp.

Capsid assembly of the two constructs was compared after co-transfectionof wildtype plasmid pTAV2.0 and codon modified plasmid pTAV/ORF1cm withempty Bluescript vector (pBS) or with pVP2N-gfp. As expected,transfection of pTAV/ORF1cm with pBS showed no detectable capsidformation, since pTAV/ORF1cm expresses all three capsid proteins butneither pTAV/ORF1 cm nor pBS express functionally active AAP. Incontrast, transfection of pTAV/ORF1 cm with pVP2N-gfp restored capsidassembly at least partially (FIG. 23C), since C-terminally truncated butactive AAP is expressed from pVP2N-gfp.

Complementation of pTAV/ORF1cm that is deficient in expression offunctional active AAP with mutant plasmids like pVP2N/ORF1cm-gfp (asdescribed in example 13) and pVP2N/ORF2stopA-gfp (see example 15) whichboth were unable to express the AAP protein (due to codon modificationor introduction of a stop codon, respectively) also did not lead tocapsid formation. In contrast, in addition to pVP2N-gfp functionallyactive AAP can be expressed from plasmids pVP2N/ORF2 cm-gfp (describedin example 13), pORF2/CTG-AU1 and pORF2/ATG-AU1 (see example 14) andrescued capsid assembly in trans-complementation (FIG. 23D).

Taken together, capsid formation in the context of the complete viralgenome is dependent on the expression of endogenous or complemented AAP.

17. Expression of Functional AAP is Necessary for Capsid Assembly in theContext of the AAV Genome

To further prove that AAP is necessary for capsid assembly in thecontext of the whole AAV genome, a stop codon was introduced in ORF2disrupting AAP amino acid sequence.

Therefore, construct pTAV/ORF2stopB was created by cloning theEcoNI/BsiWI fragment of pVP2N/ORF2stopB-gfp (for details see example 15)into the EcoNI/BsiWI backbone of pTAV2.0. (example 1.2.1). InpVP2N/ORF2stopB-gfp the caa codon starting at nucleotide was mutatedinto a taa stop codon. Hence, plasmid pTAV/ORF2stopB (schematicallyshown in FIG. 24A) encodes the known AAV2 capsid and Rep proteins butshould be deficient in the synthesis of AAP, because of the insertedstop codon.

Results and Conclusion

Again, correct expression of the four Rep proteins could be detected inWestern blot analysis (data not shown), as well as a slightly alteredexpression pattern of the three VP proteins. Expression of endogenousAAP from wildtype plasmid pTAV2.0 but not from the one containing thestop codon was directly proven using polyclonal anti-AAP serum (FIG.24B).

Capsid assembly of the two constructs was compared after co-transfectionof wildtype plasmid pTAV2.0 and mutant plasmid pTAV/ORF2stopB with emptyBluescript vector (pBS) or with pVP2N-gfp. As expected, transfection ofpTAV/ORF2stopB with pBS showed no detectable capsid formation, sincepTAV/ORF2stopB expresses all three capsid proteins but neitherpTAV/ORF2stopB nor pBS express functionally active AAP. In contrast,transfection of pTAV/ORF2stopB with pVP2N-gfp restored capsid assemblyat least partially (FIG. 24C), since C-terminally truncated but activeAAP is expressed from pVP2N-gfp.

This result further confirmed that capsid formation in the context ofthe complete viral genome is dependent on the expression of functionalAAP.

18. The “Assembly Activating Protein” AAP Targets VP Proteins to theNucleolus.

In addition to example 8, several constructs were transfected in 293-Tcells to compare the location of expressed proteins within thetransfected cell and assembly efficiency.

18.1. Cloning of Constructs

Cloning of construct pCMV-NLS-VP3 is described in example 8.1. Theapproach for generation of pCMV-NoLS-VP3 was concordant to that ofpCMV-NLS-VP3 with the difference that the complementary primer pair

(SEQ ID NO: 140) 5′-GGAAT TCGAT ATCAA GCTTG CCATG GCACG GCAGG CCCGGCGGAA TAGAC GGAGA CGGTG GCGGG AACGG CAGCG GATGG CTACA GGCAG TGG-3′, and(SEQ ID NO: 141) 5′-CCACT GCCTG TAGCC ATCCG CTGCC GTTCC CGCCA CCGTCTCCGT CTATT CCGCC GGGCC TGCCG TGCCA TGGCA AGCTT GATAT CGAAT TCC-3′was used. Accordingly, the cap gene product NoLS-VP3 contains the aminoacid sequence of the nucleolar localization signal of HIV RevMARQARRNRRRRWRERQR at the N terminus of VP3. Both constructs areschematically shown in FIG. 25A.

18.2. Analyses of Constructs by Immunofluorescence

Analogous to the experimental setup described in example 8, HeLa cellswere transfected with the different constructs as indicated. Expressionof capsid proteins and formation of capsids was analyzed byimmunofluorescence as described above using a polyclonal VP antiserum orthe monoclonal A20 antibody.

18.3. Results and Conclusion

From literature analyzing productive AAV infection (e.g. Wistuba et al.,1997) it is known that capsid assembly can first be detected in thenucleoli of infected cells. Capsid protein VP3 expressed frompCMV-VP3/2809 in HeLa cells was distributed throughout the cell nucleusand the cytoplasm and excluded from nucleoli (as shown in FIG. 11C) andno capsids were detectable in these cells upon staining with capsidspecific monoclonal antibody A20. But if AAP is co-expressed byco-transfecting pVP2N-gfp, translocation of a significant part of theVP3 protein to nucleoli and the formation of capsids could be detected.

As described in example 8, we expressed the construct pCMV-NLS-VP3 andobserved strong nuclear accumulation of VP3 fused to the nuclearlocalization signal (NLS) of SV40, which however was excluded fromnucleoli and did not cause capsid assembly (FIG. 11C). Co-expression ofAAP from plasmid pVP2N-gfp however again targeted a portion of NLS-VP3proteins to the nucleoli where capsid formation was detectable.

Interestingly, AAP protein expressed from pORF2/ATG-AU1 (described inexample 14) and stained with anti-AU1 antibody co-located withFibrillarin to the nucleoli (FIG. 25C, the phase contrast image on theright confirms location of nucleoli at the site of staining).

This result suggested that AAP co-transports VP proteins to thenucleoli, which is a prerequisite for subsequent capsid assembly.

When expressing the construct pCMV-NoLS-VP3 we observed at leastpartially nucleolar localization of VP3 fused to the nucleolarlocalization signal derived from HIV REV, but surprisingly no capsidassembly could be detected (FIG. 25B). Therefore it seemed that thetransfer of VP proteins to nucleoli is not sufficient for capsidformation. Again, co-expression of AAP from pVP2N-gfp promoted capsidformation, substantiating that AAP not only targets VP proteins to thenucleoli but plays an additional positive role in the assembly reaction.This example also shows that VP3 N-terminal insertions (I-203) aretolerated even if a highly positively charged 17mer NoLS-sequence seemsto partially interfere with VLP titers.

19. Expression of Functional AAP is Necessary for Capsid Assembly.

In addition to the immunofluorescence images seen in example 18 weanalyzed protein expression of the respective mutant constructspCMV-NLS-VP3 and pCMV-NoLS-VP3 on Western blots. Moreover, we quantifiedcapsid assembly activity of the respective constructs by monoclonalantibody A20 capsid ELISA.

Results and Conclusion

Western blot analysis confirmed expression of VP3 from pCMV-VP3/2809 andthe slightly longer proteins NLS-VP3 and NoLS-VP3 from pCMV-NLS-VP3 andpCMV-NoLS-VP3, respectively (FIG. 26A).

As already observed in example 18, neither NLS-VP3 nor NoLS-VP3 rescuecapsid formation upon cotransfection with Bluescript vector (pBS),whereas in the presence of AAP expression (from pVP2N-gfp) capsidformation was detectable (FIG. 26B).

This result confirms that AAP not only targets VP proteins to thenucleoli (which is also accomplished by the NoLS-VP3 fusion constructnot leading to capsid assembly) but also plays an essential role in theassembly reaction itself.

20. Assembly of Wildtype and VP3 VLPs

To compare the morphology of virus-like particles assembled of VP1, VP2and VP3 (VP1,2,3 VLP) with that of VLPs assembled only of VP3 (VP3 VLP)the respective samples have been investigated by electron microscopyafter negative staining using 2% uranylacetate as described above.

Virus-like particles assembled of VP1, VP2 and VP3 corresponding to thewildtype capsid were produced in 293-T cells by expression of thecomplete cap gene. VLPs assembled only of VP3 were produced byco-transfection of pCMV-VP3/2809 and pVP2N-gfp (VP3 VLP).

Results and Conclusion

Electron microscopic images confirmed that the morphology of virus-likeparticles assembled of VP1, VP2 and VP3 (VP1,2,3 VLP) is comparable tothat of VLPs assembled only of VP3 (VP3 VLP, FIG. 28). In both images,no staining of the interior is visible, therefore clearly confirmingthat all particles are empty. An image of full (DNA-containing)particles in comparison to empty particles is shown e.g. in Xie etal.(2004).

21. Trans-Complementation of AAP and VP3 Cloned from Different Serotypes

To confirm that expression of AAP from one parvovirus is capable ofmediating capsid assembly of VP3 from another parvovirus, we used therespective sequences of AAV1, AAV2 and AAV5 in trans-complementationassays.

Cloning of pVP2N-gfp of AAV1 and AAV5 was performed analogous to that ofAAV2 (compare 3.1) with the difference that primer pairs were selectedto amplify the respective sequences for AAV1 and AAV5 as given in SEQ IDNO: 24 and SEQ ID NO: 27 respectively. For trans-complementation cellswere transfected with plasmid pCMV-VP3 of either AAV2 (pCMV_VP3/2809),AAV1 (pCMV_AAV1VP3/2829) as described above or a corresponding AAV5 VP3expression construct with or without cotransfection of pVP2N-gfp of therespective AAV serotype (FIG. 29). Same molar ratios of VP3 constructand pVP2N-gfp were transfected. Particle formation efficiency wasmeasured by ELISA

Results and Conclusion

Capsid assembly of VP3 cloned from AAV1, AAV2 and AAV5, respectively,was compared after co-transfection of pVP2N-gfp cloned from AAV2 andAAV1, respectively, or Bluescript vector (pBS) (see FIG. 29). Asexpected, expression of VP3 in the absence of any other viral protein(pBS control) showed no detectable capsid formation, irrespective of itsorigin. In contrast, expression of AAP (expressed from the respectivepVP2N-gfp construct) from serotype AAV1 completely restored AAV2 VP3assembly (compared to assembly mediated by AAP from AAV2). Also vice eversa, AAP from AAV2 completely restored AAV1 VP3 assembly (compared toassembly mediated by AAP from AAV1). AAP from AAV5 was only partiallyable to complement AAV2 VP3 assembly and failed to complement AAV1 VP3assembly. Further, AAV2 and AAV1 AAP failed to complement AAV5 VP3assembly. The failure of trans-complementation with respect to AAV5constructs may be due to the fact that AAPs in these experiments werefused to GFP leading to a short C-terminal deletion of AAP which mightinterfere with the complementation of more distant parvoviruses whileactivity is sufficient for closely related serotypes. A further likelyexplanation is that more distant AAV serotypes are only partially ableto complement each other with respect to VP3 assembly. Whereas AAP fromAAV1 and AAV2 have a 71.5% identity and 81.0% similarity (Smith-WatermanAlignment), AAV2 and AAV5 only have a 56.2% identity and 60.8%similarity. These numbers are even lower with respect to AAV1 comparedto AAV5 (53.8% identity and 58.1% similarity). Accordingly, the skilledartisan will be able to select functionally active AAPs from differentserotypes and/or other functionally active variants by looking atidentities/similarities of AAP.

Still, in addition to example 11 these result confirm that parvovirusesother than AAV2 encode functional AAP and make use of the same mechanismfor capsid assembly. Further, AAP and VP3 are in principalinterchangeable between different parvoviruses, especially betweenclosely related viruses.

LITERATURE

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1-47. (canceled)
 48. A parvoviral particle consisting essentially ofVP3, i. wherein the VP3 optionally comprises one or more mutation(s),and ii. wherein the VP3 does not contain a heterologous nuclearlocalization signal, and iii. wherein the particle does not contain anyof the functional Rep proteins, particularly Rep40, Rep52, Rep68 andRep78.
 49. The parvoviral particle according to claim 48, wherein thecapsid consists only of VP3.
 50. The parvoviral particle according toclaim 48, wherein the mutation(s) of VP3 is/are: a) one or moremutation(s) selected from the group consisting of one or moredeletion(s), one or more insertion(s), one or more substitution(s), anda combination thereof; b) one or more silent mutation(s); c) one or moremutations located on the surface of a VP3 virus-like particle (VLP); d)one or more mutation(s) located at the N-terminus of VP3; e) one or moreinsertions at one or more positions selected from the group consistingof I-261, I-266, I-381, I-447, I-448, I-453, I-459, I-471, I-534, I-570,I-573, I-584, I-587, I-588, I-591, I-657, I-664, I-713, and I-716; orwherein two insertions are made at two positions selected from the groupconsisting of I-261, I-453, I-534, I-570, I-573, and I-587, whereinmutations are made into position 453 in combination with an insertion inposition 587 and in combination with an additional mutation; f) at leastone epitope heterologous to the virus, particularly wherein the epitopeof the VP3 protein is a B-cell epitope and/or particularly wherein theB-cell epitope is inserted into I-453 and/or I-587, especially intoI-453 and/or I-587 of AAV1, AAV2 or AAV4; q) wherein the VP3 is includedin a fusion protein; h) at least one tag useful for binding to a ligand;and/or i) at least one further mutation. 51-54. (canceled)
 55. Apharmaceutical composition comprising the parvoviral particle of claim48.
 56. The pharmaceutical composition of claim 55, further comprisingone or more excipients.
 57. The pharmaceutical composition of claim 55,wherein the pharmaceutical composition is a vaccine.
 58. Thepharmaceutical composition of claim 57, wherein the vaccine furthercomprises one or more adjuvants.
 59. A method for preventing or treatingan autoimmune disease, an infectious disease, a tumor disease, anallergic disease, a metabolic disease, a (chronic) inflammatory disease,a neurological disease, addiction, or an ophthalmological disease, themethod comprising administering the pharmaceutical composition of claim55 to a patient in need thereof.
 60. The method of claim 59, wherein theautoimmune disease and/or a chronic inflammatory disease is selectedfrom the group consisting of rheumatoid arthritis, psoriasis and Crohn'sdisease.
 61. The method of claim 59, wherein the tumor disease iseligible for treatment with a monoclonal antibody.
 62. The method ofclaim 59, wherein the allergic disease is selected from the groupconsisting of asthma, allergy and allergic rhinitis.
 63. The method ofclaim 59, wherein the neurological disease is Alzheimer's disease. 64.The method of claim 59, wherein the metabolic disease is atherosclerosis65. The method of claim 59, wherein the ophthalmological disease isage-related macular degeneration. 66-114. (canceled)